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Iraq Executes 18; Nine More Killed in Attacks
Wednesday: 9 Killed, 18 Executed, 37 Wounded
Attacks left nine dead and 37 wounded. The Iraqi government announced that 18 men were executed over the last couple of weeks.
The Iraqi government said that 18 Iraqi men were executed during March. The men were found guilty on terrorism-related charges.
Three people were killed and 14 more were wounded when a car bomb was detonated in Mussayab.
A bomb in Madaen left two dead and 11 wounded when it exploded.
In Mosul, two soldiers were killed and one more was wounded in an I.E.D. bombing.
A mokhtar was assassinated in Baquba.
A sticky bomb killed a police officer in Tikrit.
A car bomb targeting a police colonel's home in Dujail wounded seven people when it exploded. Among the injured were the colonel and his daughter.
In Kirkuk, a roadside bomb targeting a brigadier general in the Kurdish security forces wounded three bodyguards. One policeman was wounded in a blast targeting police dogs. A bomb targeting the provincial governor left no casualties.
Read more by Margaret Griffis
- 74 Killed in Iraq, Including Family Fleeing ISIS – July 26th, 2016
- Bomber Strike East of Baghdad; 138 Killed in Iraq – July 25th, 2016
- 164 Killed in Iraq; Bombers Strike near Shi'ite Shrine – July 24th, 2016
- 78 Killed in Iraq; Bombs Rattle Baghdad, Shirqat – July 23rd, 2016
- Sadr Militia to Fight in Mosul; 29 Killed in Iraq – July 22nd, 2016


text:
East Grand Forks News
Local news for East Grand Forks, MN continually updated from thousands of sources on the web.
East Grand Forks family left without a home after apartment fire
Monday Jul 4 | Valley News Live | Comment?
East Grand Forks news is powered by NewsRank™
Updated: Sun Jul 24, 2016 04:53 pm
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subset: commoncrawl


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Cocaine Anonymous
The following are the 12 Steps of Cocaine Anonymous. Cocaine Anonymous is a fellowship of men and women who share their experience, strength and hope with each other that they may solve their common problem and help others to recover from their addiction. The only requirement for membership is a desire to stop using cocaine and all other mind-altering substances.
For the steps below, you can click on the More about... button to read comments from books and websites about that step, or click on the People Working ... button to read about or hear people working the given step, or click on the Videos about... button to see videos about the given step. We also have forums for discussing the 12 steps in general and each of the steps in particular.
We admitted we were powerless over cocaine and all other mind-altering substances — that our lives had become unmanageable.
Came to believe that a Power greater than ourselves could restore us to sanity.
Made a decision to turn our will and our lives over to the care of God as we understood Him.
Made a searching and fearless moral inventory of ourselves.
Admitted to God, to ourselves, and to another human being the exact nature of our wrongs.
Were entirely ready to have God remove all these defects of character.
Humbly asked Him to remove our shortcomings.
Made a list of all persons we had harmed, and became willing to make amends to them all.
Made direct amends to such people wherever possible, except when to do so would injure them or others.
Continued to take personal inventory and when we were wrong promptly admitted it.
Sought through prayer and meditation to improve our conscious contact with God as we understood Him, praying only for knowledge of His will for us and the power to carry that out.
Having had a spiritual awakening as the result of these steps, we tried to carry this message to addicts, and to practice these principles in all our affairs.
Online Meetings
Calendar of online meetings
Experiences of people working the steps


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This dressing is great on a grain-based salad as well – such as quinoa with vegetables.
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I must admit that salad dressing is my big "frustration" with this program. I have come up with one salad dressing that I really do like … it's "creamy" and just "rich" enough to satisfy, plus it's a welcome change from vinegar.
I've discovered that I really like raspberry dressing, but most of the fatfree ones in the grocery store have too much sugar. I found this recipe and have been experimenting with cutting down on the calories by decreasing the apple juice concentrate.
This is my lower-fat, vegan version of Annie's Gingerly Vinaigrette (which contains honey). I've used ground flax seeds to thicken it–as well as add essential Omega 3's.
Good on salad, avocado or jacket potatoes. You may want to add seasonings to taste.
4 tsp. Ground coriander
1 tsp. Pepper…
1 T tamari soy sauce
1 teas dark sesame oil…
¼ cup creamy peanut butter
¼ cup firmly packed brown sugar…
1/2 cup TVP granules
1 tablespoon liquid smoke flavoring — (1 to 2 tablespoons)…
This is the sauce used in my Pasta Primavera recipe. In addition to being great with pasta, it makes a delicious sauce for steamed vegetables or baked potatoes–or both! –SusanV
4 cups seeded and peeled watermelon — 1/2-inch cubes
1/2 cup diced red onion…
1 medium red or white onion, peeled and diced
[salt] and pepper to taste…
This is a great way to use up zucchini if your garden provides a little too much in the summer months!
1 can green beans, drained, 16 oz.
1 can wax beans, drained, 16 oz….
8 ounces baked tofu
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Skip to comments.Obama Wins Georgia Ballot Challenge (Born in the USA Qualifies as a Natural Born Citizen)
Posted on 02/04/2012 5:43:26 AM PST by broken_arrow1
President Obama's name should appear on Georgia's 2012 presidential ballot, in the official opinion of Judge Michael Malihi of Georgia's Office of State Administrative Hearings (OSAH), issued on February 3. Judge Malihi's decision is the result of hearings held January 26 on three separate actions brought by several Georgia residents. Under Georgia law, Secretary of State Brian Kemp had referred the challenges, filed last November, to the OSAH for a recommendation.
Ordinarily, the Court would enter a default order against the party that fails to participate in any stage of a proceeding...Nonetheless, despite the Defendant's failure to appear, Plaintiffs asked this Court to decide the case on the merits of their arguments and evidence. The Court granted Plaintiff's request.
Two of the challenges, represented by attorney Van Irion of the Liberty Legal Foundation and Georgia Rep. J. Mark Hatfield, did not focus on Obama's place of birth or the infamous birth certificate. Rather, Irion and Hatfield contended that Obama, with his non-US citizen father, is not a "natural born" citizen according to the rule of statutory construction in the interpretation of the Constitution and existing Supreme Court precedent. (Further explanation of those assertions is contained in a comprehensive amicus brief submitted to the court, prepared by attorney Leo Donofrio.)
In his sweeping denial of the Plaintiff's challenges, however, Judge Malihi did not mention the principle, and instead relied on the 2009 case of Ankeny v Governor, stating that "[t]he Indiana Court determined that a person qualifies as a natural born citizen if he was born in the United States because he became a United States citizen at birth."
(Excerpt) Read more at americanthinker.com ...
So now we can now run Jindal 2016 since 0 is definitely retaining.
Rubio is in.
The Chicago mob showed up at the Judge's home.
The "fix" is in also!!!
Obozo was hatched under a rock; the progeny of two mating scorpions.
The judge was made an offer he couldn't refuse.
This decision is SO bogus!
Becoming a 'United States citizen at birth' means naturalized AT birth.
It has nothing to do with being natural-born. Natural born citizenship can only be bestowed by Natural Law, not positive law.
It's why the Founders called it NATURAL BORN!
Did anyone really expect any other outcome? It doesn't matter what this guy has or hasn't done (which most of us will never know i.e., school, ex-girl/boy friend, SS#,why birth certificate says African for his father(?) etc,etc) nobody will step up and really challenge him out of fear of being called a racist. Last best hope is November. Pray the wool is pulled from over the sheeples eyes by then. Danger Will Robinson, Danger!!!!
So this means an anchor baby could be President.
"The judge was made an offer he couldnt refuse"
This was ABSOLUTELY what happened.
The union is splitting ala the Cival War ... another war between states.
How does a judge determine Obama was "born in the USA" when he was presented with evidence of a FRAUDULENT birth record??
I'm going to clear out of hear even sooner than I thought I would.
America has left me more than I am leaving America.
The corruption has become so thick that you can't cut through it with an axe.
Someone needs to point out to Lindsey Graham that this decision means anchor babies can run for president. Maybe that'll get his attention.
Rubio is in.
No and no!
Why should we descend to their level. I am sick of the continual erosion of the Constitution and our liberties. Our Constitution should be defended much like the U.S. Army Rangers who assailed the 100 meter high cliffs at Pointe du Hoc on D-Day defended it.
Before you know it, anchor babies will be running.
But wait...Bill Richardson, an anchor baby, has tried to run with no repercussions.
They are transforming America, thank god we have Romney.............I'm sptiing up right now
The judge wimped out. He has ruled in such a manner as to punt the ball to the Georgia Superior Court and then the SCOTUS. Hatfield certainly will be filing an appeal and I would expect Irion to do the same.
One knew this was going to happen. Bowbamaâs Chicago people surely intervened. Just when I thought there is justice in jurisprudence, I/we get kicked in the nads as the Constitution dies!
So if your choice was Rubio or Obama, would you not vote?
So this means an anchor baby could be President.
"So would have been Anwar al-Awlaki." That's the terrorist that was a US citizen that Obama had assassinated.
The fix was in long before the trial ever came up.
Obama knew it and his lawyer knew it. Neither even bothered to show up in court.
At the very least they were guilty of contempt of court, but you won't be seing that charge come up.
This was all a farce by the Judge who got his name in the papers.
What is more remarkable is that a photoshop document is considered evidence of Hawaii birth. Obama committed fraud on the court and yet wins the case. This is ground breaking.
I doubt that. Anybody who believed, even for a second, that any judge in any courtroom in this country was going to embrace any of this this birther nonsense is an idiot.
If he had a snowball's chance, he might have been investigated more closely. :-)
It just gets me they try to pin all this on the Wong Kim Ark case.
Not only did Wong Kim petition the court as a NATIVE [not natural born] citizen, the judge said this in his decision:
The right of citizenship never descends in the legal sense, either by the common law or under the common naturalization acts. It is incident to birth in the country, or it is given personally by statute. The child of an alien, if born in the country, is as much a citizen as the natural born child of a citizen, and by operation of the same principle.
If there were no difference, the judge would have stated no distinction.
It's like saying a car is just as much a vehicle as a truck. A true statement in itself, but it doesn't make a car INTO a truck!
I guess I just don't understand why do many folks have trouble comprehending this.
[Not that YOU do, of course....but thanks for the opportunity to rant. LOL!]
Article II, Section 1, Clause 5.
Neither should be on the ballot.
Not the news we we looking for; we better hope for change!
Reminds me of the Clinton/Whitewater/Lewinsky days. Big build ups to supposedly catastrophic events that would wash Clinton from the Whitehouse.
Never happened and the Dems went away laughing.
Did you watch the hearing?
Because the majority of the hearing had nothing to do with nut job Orly Taitz...they should have never allowed in there.
She gave the judge the perfect out and route of deflection.
I watched the entire hearing and the evidence presented in the beginning was excellent.
However for the life of me I don't understand why they didn't take the summary judgment. They had it won and they blew it...what a bunch of fools!!!
Unfortunately, it only seems to be important to those of us who care.
Send our regards to Prince Prospero!
I suspect between rural and urban areas. Friend of mine said his relationships with friends of different races have really soured. And those friendships were long standing.
Prince Prospero?
Sorry, never heard of him.
Please explain.
Ark has nothing to do with it.
Only somebody grasping at straws would even bring it up.
Now what grounds for an appeal could there possibly be?
Everybody knows the Founding Fathers intended for a single Indiana Judge to have the final say in the matters of the constitution and lord over all the other states.
The judge decided the case based on current law, because that is what the judge has to do.
Like it or not, believe in the natural born citizen interpretation at the time of the Founders or not, the ruling piece of paper is going to be current law.
The work that has be done for the future is changing that law to reflect original intent, and that hasn't been done, and it won't be done with this Congress (or the next).
Imho, there's no way that Jindal qualifies. Also, imho, Rubio does if one uses the citizenship law of 1792 for his father.
Is PP a FReeper? I never heard of one by that name.
If you are talking about the long-dead prince...well, I'm planning to leave, not to kill myself.
What evidence did the defendants present to the court that disputed her case?
"The Masque of the Red Death" (1842), is a short story by Edgar Allan Poe. The story follows Prince Prospero's attempts to avoid a dangerous plague known as the Red Death by hiding in his abbey. He, along with many other wealthy nobles, has a masquerade ball within seven rooms of his abbey, each decorated with a different color. In the midst of their revelry, a mysterious figure enters and makes his way through each of the rooms. Prospero dies after confronting this stranger, as do the guests."
Maybe there is more to your story than I realize, but Prospero comes to mind whenever someone informs of their plans to escape America. In reality America is just starting to get exciting after a long, dull period!
Yup, I'm thinking someone had a educational chat with the good judge. And I'm also thinking all hell is about to break out in the US.
Every member of the Supreme Court, every member of congress, every member of the Joint Chiefs, most members of the DOD, CIA, FBI, Secret Service and state run media, ABC, CBS, NBC, CNN, PBS, NPR, MSNBC, Fox and print news, knows that Barack Hussein Obama does NOT meet Article II Section I constitutional requirements for the office he holds. By his own biography, there is NO way he can pass the test. The hard evidence is so far beyond overwhelming, it is ridiculous.
But not ONE member of Americas most powerful people will dare confront Obama and his anti-American cabal on the subject. The Constitution does NOT stand.
Half of the people you expect to stop this insanity are quiet co-conspirators in the silent coup. The other half is paralyzed by fear, motivated only by political self-preservation.
Americans keep asking what they can do because they see that none of their leaders are doing anything to stop the demise of their beloved country. Its the right question, because those leaders are NOT going to stop this thing.
Who will save freedom?
A brave few
This is how it was in the beginning, how it has always been and how it will be.
Dr. Orly Taitz, Phil Berg and Gary kreep, all of whom have made defending the constitution and the American way of life a personal ambition in the absence of any constitutional leadership.
If the American people fail to get behind these brave few who are seeking peaceful redress, all the peaceful options will evaporate as if they never existed. We will return to a pre-1776 America overnight.
no one will challenge him out of more fear than that
I vote civil war in that case, and anyone who supported this is a combatant and not to be accorded quarter.
I once swore to defend the constitution against all enemies foreign and domestic. No passes to enemies who are merely idiots.
You nuts have screwed everyone.
I would stay home, no voting for any anchor baby, not ever.
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Tour de France 2009 Hits the XBLA Today
Written Wednesday, July 15, 2009 By Dan WebbView author's profile
In a surprise release today, as well as Monkey Island & Madballs in ... Babo: Invasion being released on the Xbox Live Arcade, CYANIDE's Tour de France 2009 is available as well.
CYANIDE's Tour de France 2009 - The Official Game, allows you to choose one of the 18 official teams and lead it through the 21 official Tour de France 2009 legs. Early breakaways, fights for jerseys and sprints are some of the possible behaviours that are particularly specific to the sport. You can even take the action on to Xbox Live with 4 player action confirmed.
All three games are available now for 800 Microsoft Points each.


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Is there a better diplomat in boxing than Oscar De La Hoya? Days after promoter Bob Arum stopped coughing up mold spores long enough to weigh in on the UFC, the mostly retired boxer gave The Orange County Register a fair assessment of his event's chances against UFC 103 on Saturday. (De La Hoya is co-promoting the Floyd Mayweather-Juan Manuel Marquez bout, which airs opposite the UFC offering of Vitor Belfort versus Rich Franklin.)
"We have no problem with [going up against the UFC]," De La Hoya said. "Look, we have different audiences. We have our own marketing plan. They have their own marketing plan. We're almost sold out. I don't know about them, but Look, we have the '24/7' thing [on HBO] and tremendous sponsors behind us. They have great sponsors behind them. We're in our own worlds. Let them do great, we're gonna do great and everybody wins."
Are their audiences really so different? Although boxing fans often can skew older, there's undoubtedly an overlap -- and where those viewers lean will influence who earns bragging rights for the weekend. The last shows topped by Franklin, UFCs 93 and 99, sold an estimated 320,000-360,000 buys but were hampered by a notoriously fickle attitude from fans about tape-delayed UK events; Mayweather's last fight, versus UK star Ricky Hatton, sold 925,000 buys domestically. Mayweather's bout with De La Hoya scored 2.4 million, an industry record. But Mayweather's fight against a little-known Carlos Baldomir netted only 325,000 homes.
It will be a very interesting weekend.


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New Belly Fat Weight Loss Exercise Takes Less Time and Works Better, Say Researchers
Researchers from the School of Medical Sciences at the University of New South Wales have recently published their development of a new type of exercise called "LifeSprints" that they say causes significant loss of belly fat in a relatively short time in comparison to other types of exercise that take longer to achieve the same amount of belly fat weight loss.
Funded by Diabetes Australia, LifeSprints was developed from research conducted at the University of New South Wales to develop innovative and effective exercises for weight loss. Basically, LifeSprints research has demonstrated that a 20-minute workout consisting of 8 seconds of sprint exercise, followed by 12 seconds of relative recovery resulted in more fat loss than 40 minutes of steady state exercise. The fast-paced LifeSprints style of exercise includes the use of cycling, boxing and equipment like elliptical fitness trainers found in fitness centers.
In the latest issue of the Journal of Obesity, researchers report their findings that obese men utilizing the LifeSprints style of interval sprinting on stationary bicycles lost in one hour what normally takes 7 hours of jogging to achieve in weight loss. According to the researchers, just 20 minutes of doing sprints on an exercise bike, three times a week, is all that is needed to achieve significant weight loss around the abdomen.
"Sprints are a very time efficient form of exercise," says Associate Professor Steve Boutcher, who led the UNSW Medicine research. "The sprint program, LifeSprints, reduced visceral fat with seven times less exercise time and has a much greater impact on cardiovascular and metabolic health than reductions of subcutaneous fat stores in the legs and arms."


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Turkish Chickpea & Lamb Soup
From EatingWell: March/April 2010
This recipe was inspired by a hearty lamb and chickpea soup from Turkey. Finishing it with a dollop of pistachio-mint pesto adds another layer of flavor.
Makes: 6 servings, about 1 2/3 cups each
Active Time:
Total Time:
Nutrition Profile
Healthy weight | High potassium | Low cholesterol | Low saturated fat | High fiber | Low calorie | Heart healthy | Diabetes appropriate |View Our Nutrition Guidelines »
- 3/4 cup dried chickpeas, picked over and rinsed
- 3 teaspoons extra-virgin olive oil, divided
- 1 large onion, chopped
- 2 medium carrots, diced
- 3 cloves garlic, minced
- 2 teaspoons ground cumin
- 1 teaspoon ground coriander
- 1/4 teaspoon cayenne pepper
- 1 1-pound lamb shank, trimmed
- 1/8 teaspoon salt
- 1/4 teaspoon freshly ground pepper
- 4 cups reduced-sodium chicken broth
- 1 14-ounce can diced tomatoes
- 2 bay leaves
- 1/2 cup bulgur (see Note), rinsed
- 2 tablespoons lemon juice
Pistachio-Mint Pesto
- 1 1/2 cups fresh mint leaves
- 1/3 cup shelled pistachios
- 2 cloves garlic, crushed and peeled
- 2 tablespoons extra-virgin olive oil
- 2 tablespoons lemon juice
- 2 tablespoons low-fat plain yogurt
- 1/8 teaspoon salt (optional)
- To prepare soup: Place chickpeas in a large bowl. Add enough cold water to cover by 3 inches and let soak for 8 to 24 hours. (Alternatively, place chickpeas in a large saucepan and cover with 3 inches cold water. Bring to a boil and cook for 2 minutes. Remove from the heat. Let stand for 1 hour.) Drain.
- Heat 2 teaspoons oil in 4-quart or larger pressure cooker over medium-high heat. Add onion and carrots; cook, stirring, until softened, 3 to 5 minutes. Add garlic, cumin, coriander and cayenne; cook, stirring, until fragrant, about 20 seconds. Remove from the heat.
- Sprinkle lamb shank with salt and pepper. Heat the remaining 1 teaspoon oil in a medium skillet, preferably cast-iron, over medium heat. Add the lamb shank and cook, turning from time to time, until browned all over, 3 to 7 minutes. Transfer to the pressure cooker. Add broth, tomatoes, bay leaves and the chickpeas.
- Secure the pressure cooker lid. Bring to high pressure over high heat following manufacturer's directions. Reduce the heat to the lowest setting that maintains high pressure. Cook for 35 minutes.
- Meanwhile, place bulgur in a medium bowl. Cover with boiling water and let stand for 15 minutes. Drain in a sieve, pressing out excess water.
- Let the pressure release naturally. This will take 5 to 20 minutes.
- To prepare pesto: Meanwhile, process mint, pistachios and garlic in a food processor until finely chopped. With the motor running, gradually add oil, lemon juice and yogurt through the feed tube. Process until the mixture forms a paste. If using unsalted pistachios, add salt, if desired.
- When the pressure has released, discard bay leaves. Skim fat from the surface of the soup. Using tongs, transfer the lamb shank to a clean cutting board. When cool enough to handle, coarsely shred the lamb meat and return it to the soup. Add the drained bulgur. Heat through. Stir in lemon juice. Dollop some pesto onto each serving.
Tips & Notes
- Make Ahead Tip: Cover and refrigerate for up to 2 days. Thin with additional chicken broth or water, if necessary, when reheating. | Equipment: 4-quart or larger pressure cooker
- Note: Bulgur is made by parboiling, drying and coarsely grinding or cracking wheat berries. Don't confuse bulgur with cracked wheat, which is just that—cracked wheat. Since the parboiling step is skipped, cracked wheat must be cooked for up to an hour whereas bulgur simply needs a quick soak in hot water for most uses. Look for it in the natural-foods section of large supermarkets, near other grains.
- High-Altitude Tip: For every 1,000 feet above 2,000 feet elevation, increase the cooking time by 5%.
- Conventional-Stove Variation: In Step 2, replace the pressure cooker with a 4- to 6-quart Dutch oven. After sautéing onion, carrots, garlic, cumin, coriander and cayenne, transfer to a plate. Brown the lamb shank (Step 3) in the same pot. Add onion and carrot mixture back to the pot along with the chickpeas. In Step 4, simmer gently, covered, over low heat, until the lamb shank and chickpeas are very tender, 1 3/4 to 2 1/4 hours.
Per serving: 329 calories; 13 g fat (2 g sat, 8 g mono); 21 mg cholesterol; 37 g carbohydrates; 0 g added sugars; 18 g protein; 10 g fiber; 664 mg sodium; 748 mg potassium.
Nutrition Bonus: Vitamin A (95% daily value), Vitamin C (40% dv), Iron (33% dv), Potassium (21% dv), Zinc (18% dv)
Carbohydrate Servings: 2
Exchanges: 1 1/2 starch, 1 vegetable, 1 1/2 lean meat, 2 fat
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Recipe Categories
- Meal/Course
- Dinner
- Health & Diet Considerations
- Healthy weight
- High fiber
- High potassium
- Diabetes appropriate
- Low calorie
- Low cholesterol
- Low saturated fat
- Heart healthy
- Style/Theme
- Budget
- Entertaining, casual
- Make ahead instructions
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- Main dish, meat
- Soups/stews
- Ethnic/Regional
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- Servings
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- Main Ingredient
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- Publication
- March/April 2010


text:
Summer Season (1985– )
Episode List
11 Jun. 1985Long Term Memory
13 Jun. 1985Rachel and the Roarettes
18 Jun. 1985Phoebe
20 Jun. 1985You've Never Slept in Mine
25 Jun. 1985Paris
Two old ladies meet in Glasgow and weave fantasies about trips to Paris.
2 Jul. 1985Picture Friend
After a drunken woman falls off a bus, and is taken to hospital overnight, she starts to have disturbing memories.
4 Jul. 1985Pity in History
9 Jul. 1985Glamour Night
The play involves a glamour photography session. During the photo-shoot the photographers begin trying to coerce a model into posing topless, leading eventually to an angry outburst from the model.
11 Jul. 1985Time Trouble
Tension between the East and the West escalate and the radio warns that a nuclear war is becoming a distinct possibility. Liverpudlian Michael Angelis takes his two young sons to London by train. The older of the two boys, Carl, is a chess prodigy. The younger son is a normal boy with no interest in chess. They arrive at a Chess event where visiting Russian Grand Master Krylov plays several games simultaneously. Unbeknown to his accompanying KGB escort Krylov is hoping to manufacture an opportunity in which he can defect. Krylov easily beats all of his opponents ...
16 Jul. 1985Radio Pictures
Actors rehearse and record a radio play.
18 Jul. 1985Maybe Baby
23 Jul. 1985The Dumb Waiter
25 Jul. 1985One for the Road
30 Jul. 1985Pythons on the Mountain
1 Aug. 1985A Still Small Shout
6 Aug. 1985A Crack in the Ice
8 Aug. 1985Flights
13 Aug. 1985Tuesday's Child
15 Aug. 1985Urban Jungle
20 Aug. 1985Broken Homes
22 Aug. 1985Kisses on the Bottom
What do the characters on the seaside postcards get up to in private?
27 Aug. 1985The House on Kirov Street
29 Aug. 1985After You, Hugo
3 Sep. 1985Portrait of Isa Mulvenny
5 Sep. 1985Reservations


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Johansen, Lisa M
Molecular Systems Biology
SREBP-2, oxidosqualene cyclase (OSC) or lanosterol demethylase were identified as novel sterol pathway-associated targets that, when probed with chemical agents, can inhibit hepatitis C virus (HCV) replication.Using a combination chemical genetics approach, combinations of chemicals targeting sterol pathway enzymes downstream of and including OSC or protein geranylgeranyl transferase I (PGGT) produce robust and selective synergistic inhibition of HCV replication. Inhibition of enzymes upstream of OSC elicit proviral responses that are dominant to the effects of inhibiting all downstream targets.Inhibition of the sterol pathway without inhibition of regulatory feedback mechanisms ultimately results in an increase in HCV replication because of a compensatory upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression. Increases in HMGCR expression without inhibition of HMGCR enzymatic activity ultimately stimulate HCV replication through increasing the cellular pool of geranylgeranyl pyrophosphate (GGPP).Chemical inhibitors that ultimately prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced.
Chemical inhibition of enzymes in either the cholesterol or the fatty acid biosynthetic pathways has been shown to impact viral replication, both positively and negatively (Su et al, 2002; Ye et al, 2003; Kapadia and Chisari, 2005; Sagan et al, 2006; Amemiya et al, 2008). FBL2 has been identified as a 50 kDa geranylgeranylated host protein that is necessary for localization of the hepatitis C virus (HCV) replication complex to the membranous web through its close association with the HCV protein NS5A and is critical for HCV replication (Wang et al, 2005). Inhibition of the protein geranylgeranyl transferase I (PGGT), an enzyme that transfers geranylgeranyl pyrophosphate (GGPP) to cellular proteins such as FBL2 for the purpose of membrane anchoring, negatively impacts HCV replication (Ye et al, 2003). Conversely, chemical agents that increase intracellular GGPP concentrations promote viral replication (Kapadia and Chisari, 2005). Statin compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the sterol pathway (Goldstein and Brown, 1990), have been suggested to inhibit HCV replication through ultimately reducing the cellular pool of GGPP (Ye et al, 2003; Kapadia and Chisari, 2005; Ikeda et al, 2006). However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. The use of statins for the treatment of HCV is likely to be complicated by the reported compensatory increase in HMGCR expression in vitro and in vivo (Stone et al, 1989; Cohen et al, 1993) in response to treatment. Enzymes in the sterol pathway are regulated on a transcriptional level by sterol regulatory element-binding proteins (SREBPs), specifically SREBP-2 (Hua et al, 1993; Brown and Goldstein, 1997). When cholesterol stores in cells are depleted, SREBP-2 activates transcription of genes in the sterol pathway such as HMGCR, HMG-CoA synthase, farnesyl pyrophosphate (FPP) synthase, squalene synthase (SQLS) and the LDL receptor (Smith et al, 1988, 1990; Sakai et al, 1996; Brown and Goldstein, 1999; Horton et al, 2002). The requirement of additional downstream sterol pathway metabolites for HCV replication has not been completely elucidated.
To further understand the impact of the sterol pathway and its regulation on HCV replication, we conducted a high-throughput combination chemical genetic screen using 16 chemical probes that are known to modulate the activity of target enzymes relating to the sterol biosynthesis pathway (Figure 1). Using this approach, we identified several novel antiviral targets including SREBP-2 as well as targets downstream of HMGCR in the sterol pathway such as oxidosqualene cyclase (OSC) and lanosterol demethylase. Many of our chemical probes, specifically SR-12813, farnesol and squalestatin, strongly promoted replicon replication. The actions of both farnesol and squalestatin ultimately result in an increase in the cellular pool of GGPP, which is known to increase HCV replication (Ye et al, 2003; Kapadia and Chisari, 2005; Wang et al, 2005).
Chemical combinations targeting enzymes upstream of squalene epoxidase (SQLE) at the top of the sterol pathway (Figure 4A) elicited Bateson-type epistatic responses (Boone et al, 2007), where the upstream agent's response predominates over the effects of inhibiting all downstream targets. This was especially notable for combinations including simvastatin and either U18666A or squalestatin, and for squalestatin in combination with Ro48-8071. Treatment with squalestatin prevents the SQLS substrate, farnesyl pyrophosphate (FPP) from being further metabolized by the sterol pathway. As FPP concentrations increase, the metabolite can be shunted away from the sterol pathway toward farnesylation and GGPP synthetic pathways, resulting in an increase in host protein geranylgeranylation, including FBL2, and consequently replicon replication. This increase in replicon replication explains the source of the observed epistasis over Ro48-8071 treatment.
Combinations between probes targeting enzymes downstream of and including OSC produced robust synergies with each other or with a PGGT inhibitor. Figure 4B highlights examples of antiviral synergy resulting from treatment of cells with an OSC inhibitor in combination with an inhibitor of either an enzyme upstream or downstream of OSC. A combination of terconazole and U18666A is synergistic without similar combination effects in the host proliferation screen. Likewise, clomiphene was also synergistic when added to replicon cells in combination with U18666A. One of the greatest synergies observed downstream in the sterol pathway is a combination of amorolfine and AY 9944, suggesting that there is value in developing combinations of drugs that target enzymes in the sterol pathway, which are downstream of HMGCR.
Interactions with the protein prenylation pathway also showed strong mechanistic patterns (Figure 4C). GGTI-286 is a peptidomimetic compound resembling the CAAX domain of a protein to be geranylgeranylated and is a competitive inhibitor of protein geranylgeranylation. Simvastatin impedes the antiviral effect of GGTI-286 at low concentrations but that antagonism is balanced by comparable synergy at higher concentrations. At the low simvastatin concentrations, a compensatory increase in HMGCR expression leads to increased cellular levels of GGPP, which are likely to result in an increase in PGGT enzymatic turnover and decreased GGTI-286 efficacy. The antiviral synergy observed at the higher inhibitor concentrations is likely nonspecific as synergy was also observed in a host viability assay. Further downstream, however, a competitive interaction was observed between GGTI-286 and squalestatin, where the opposing effect of one compound obscures the other compound's effect. This competitive relationship between GGTI and SQLE explains the epistatic response observed between those two agents. For inhibitors of targets downstream of OSC, such as amorolfine, there are strong antiviral synergies with GGTI-286. Notably, combinations with OSC inhibitors and GGTI-286 were selective, in that comparable synergy was not found in a host viability assay. This selectivity suggests that jointly targeting OSC and PGGT is a promising avenue for future HCV therapy development.
This study provides a comprehensive and unique perspective into the impact of sterol pathway regulation on HCV replication and provides compelling insight into the use of chemical combinations to maximize antiviral effects while minimizing proviral consequences. Our results suggest that HCV therapeutics developed against sterol pathway targets must consider the impact on underlying sterol pathway regulation. We found combinations of inhibitors of the lower part of the sterol pathway that are effective and synergistic with each other when tested in combination. Furthermore, the combination effects observed with simvastatin suggest that, though statins inhibit HMGCR activity, the resulting regulatory consequences of such inhibition ultimately lead to undesirable epistatic effects. Inhibitors that prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced.
The search for effective Hepatitis C antiviral therapies has recently focused on host sterol metabolism and protein prenylation pathways that indirectly affect viral replication. However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. Here, we present a combination chemical genetic study to explore how the sterol and protein prenylation pathways work together to affect hepatitis C viral replication in a replicon assay. In addition to finding novel targets affecting viral replication, our data suggest that the viral replication is strongly affected by sterol pathway regulation. There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway. We also show how pathway regulation frustrates potential hepatitis C therapies based on the sterol pathway, and reveal novel synergies that selectively inhibit hepatitis C replication over host toxicity. In particular, combinations targeting the downstream sterol pathway enzymes produced robust and selective synergistic inhibition of hepatitis C replication. Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.
chemical genetics; combinations and synergy; hepatitis C; replicon; sterol biosynthesis
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Rab GTPase Prenylation Hierarchy and Its Potential Role in Choroideremia Disease
Köhnke, Monika
Delon, Christine
Hastie, Marcus L.
Nguyen, Uyen T. T.
Wu, Yao-Wen
Waldmann, Herbert
Goody, Roger S.
Gorman, Jeffrey J.
Alexandrov, Kirill
PLoS ONE
Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.
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Membrane Targeting of Rab GTPases Is Influenced by the Prenylation MotifD⃞
Gomes, Anita Q.
Ali, Bassam R.
Ramalho, José S.
Godfrey, Richard F.
Barral, Duarte C.
Hume, Alistair N.
Seabra, Miguel C.
Molecular Biology of the Cell
Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27aash) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIα and propose that the mistargeting of Rabs to the ER results from loss of targeting information.
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Molecular Basis for Rab Prenylation
Alory, Christelle
Balch, William E.
The Journal of Cell Biology
Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.
choroideremia; REP1; CHM; vesicle traffic; MRS6
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The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
Overmeyer, Jean H.
Wilson, Amy L.
Erdman, Robert A.
Maltese, William A.
Molecular Biology of the Cell
Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.
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Use of Synthetic Isoprenoids to Target Protein Prenylation and Rho GTPases in Breast Cancer Invasion
Chen, Min
Knifley, Teresa
Subramanian, Thangaiah
Spielmann, H. Peter
O'Connor, Kathleen L.
PLoS ONE
Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.
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Rab GDI: a solubilizing and recycling factor for rab9 protein.
Soldati, T
Riederer, M A
Pfeffer, S R
Molecular Biology of the Cell
Rab proteins are thought to function in the processes by which transport vesicles identify and/or fuse with their respective target membranes. The bulk of these proteins are membrane associated, but a measurable fraction can be found in the cytosol. The cytosolic forms of rab3A, rab11, and Sec4 occur as equimolar complexes with a class of proteins termed "GDIs," or "GDP dissociation inhibitors." We show here that the cytosolic form of rab9, a protein required for transport between late endosomes and the trans Golgi network, also occurs as a complex with a GDI-like protein, with an apparent mass of approximately 80 kD. Complex formation could be reconstituted in vitro using recombinant rab9 protein, cytosol, ATP, and geranylgeranyl diphosphate, and was shown to require an intact rab9 carboxy terminus, as well as rab9 geranylgeranylation. Monoprenylation was sufficient for complex formation because a mutant rab9 protein bearing the carboxy terminal sequence, CLLL, was prenylated in vitro by geranylgeranyl transferase I and was efficiently incorporated into 80-kD complexes. Purified, prenylated rab9 could also assemble into 80-kD complexes by addition of purified, rab3A GDI. Finally, rab3A-GDI had the capacity to solubilize rab9GDP, but not rab9GTP, from cytoplasmic membranes. These findings support the proposal that GDI proteins serve to recycle rab proteins from their target membranes after completion of a rab protein-mediated, catalytic cycle. Thus GDI proteins have the potential to regulate the availability of specific intracellular transport factors.
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Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.
Sanford, J C
Pan, Y
Wessling-Resnick, M
Molecular Biology of the Cell
Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.
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Towards Complete Sets of Farnesylated and Geranylgeranylated Proteins
Maurer-Stroh, Sebastian
Koranda, Manfred
Benetka, Wolfgang
Schneider, Georg
Sirota, Fernanda L
Eisenhaber, Frank
PLoS Computational Biology
Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein–protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins—for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain–containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.
Author Summary
Various cellular functions require reversible membrane localization of proteins. This is often facilitated by attaching lipids to the respective proteins, thus anchoring them to the membrane. For example, addition of prenyl lipid anchors (prenylation) is directed by a motif in the protein sequence that can be predicted using a recently developed method. We describe the prediction of protein prenylation in all currently known proteins. The annotated results are available as an online database: PRENbase. A ranking of the predictions is introduced, assuming that existence of a prenylation sequence motif in related proteins from different species (evolutionary conservation) relates to functional importance of the lipid anchor. We present experimental evidence for high-ranked human proteins predicted to be affected by anticancer drugs inhibiting prenylation.
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Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization*
Berg, Tracy J.
Gastonguay, Adam J.
Lorimer, Ellen L.
Kuhnmuench, John R.
Li, Rongshan
Fields, Alan P.
Williams, Carol L.
The Journal of Biological Chemistry
Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.
Low Molecular Weight G Proteins; Protein Isoprenylation; Ras; Rho; Trafficking; Rac; Rap; Rap1GDS1; SmgGDS; Polybasic
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Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions
Okamoto, S
Jiang, Y
Kawamura, K
Shingyoji, M
Tada, Y
Sekine, I
Takiguchi, Y
Tatsumi, K
Kobayashi, H
Shimada, H
Hiroshima, K
Tagawa, M
Cell Death & Disease
Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.
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Dual Prenylation Is Required for Rab Protein Localization and Function
Calero, Monica
Chen, Catherine Z.
Zhu, Wenyan
Winand, Nena
Havas, Karyn A.
Gilbert, Penny M.
Burd, Christopher G.
Collins, Ruth N.
Molecular Biology of the Cell
The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.
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Impaired prenylation of Rab GTPases in the gunmetal mouse causes defects in bone cell function
Taylor, Adam
Mules, Emilie H
Seabra, Miguel C
Helfrich, Miep H
Rogers, Michael J
Coxon, Fraser P
Small GTPases
Vesicular trafficking is crucial for bone resorption by osteoclasts, in particular for formation of the ruffled border membrane and for removal of the resultant bone degradation products by transcytosis. These processes are regulated by Rab family GTPases, whose activity is dependent on post-translational prenylation by Rab geranylgeranyl transferase (RGGT). Specific pharmacological inhibition of RGGT inhibits bone resorption in vitro and in vivo, illustrating the importance of Rab prenylation for osteoclast function. The gunmetal (gm/gm) mouse bears a mutation in the catalytic subunit of RGGT, causing a loss of 75% of the activity of this enzyme and hence hypoprenylation of several Rabs in melanocytes, platelets and cytotoxic T cells. We have now found that prenylation of several Rab proteins is also defective in gm/gm osteoclasts. Moreover, while osteoclast formation and cytoskeletal polarization occurs normally, gm/gm osteoclasts exhibit a substantial reduction in resorptive activity in vitro compared with osteoclasts from +/gm mice, which do not have a prenylation defect. Surprisingly, rather than the osteosclerosis that would be expected to result from defective osteoclast function in vivo, gm/gm mice exhibited a slightly lower bone mass than +/gm mice, indicating that defects in other cell types, such as osteoblasts, in which hypoprenylation of Rabs was also detected, may contribute to the phenotype. However, gm/gm mice were partially protected from ovariectomy-induced bone loss, suggesting that levels of Rab prenylation in gm/gm osteoclasts may be sufficient to maintain normal physiological levels of activity, but not pathological levels of bone resorption in vivo.
osteoclast; bone resorption; bone; Rab; small GTPase; prenylation; gunmetal
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Statin Treatment Increases Lifespan and Improves Cardiac Health in Drosophila by Decreasing Specific Protein Prenylation
Spindler, Stephen R.
Li, Rui
Dhahbi, Joseph M.
Yamakawa, Amy
Mote, Patricia
Bodmer, Rolf
Ocorr, Karen
Williams, Renee T.
Wang, Yinsheng
Ablao, Kenneth P.
PLoS ONE
Statins such as simvastatin are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and standard therapy for the prevention and treatment of cardiovascular diseases in mammals. Here we show that simvastatin significantly increased the mean and maximum lifespan of Drosophila melanogaster (Drosophila) and enhanced cardiac function in aging flies by significantly reducing heart arrhythmias and increasing the contraction proportion of the contraction/relaxation cycle. These results appeared independent of internal changes in ubiquinone or juvenile hormone levels. Rather, they appeared to involve decreased protein prenylation. Simvastatin decreased the membrane association (prenylation) of specific small Ras GTPases in mice. Both farnesyl (L744832) and type 1 geranylgeranyl transferase (GGTI-298) inhibitors increased Drosophila lifespan. These data are the most direct evidence to date that decreased protein prenylation can increase cardiac health and lifespan in any metazoan species, and may explain the pleiotropic (non-cholesterol related) health effects of statins.
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The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.
Beranger, F
Paterson, H
Powers, S
de Gunzburg, J
Hancock, J F
Molecular and Cellular Biology
C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.
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Rab27a Is Required for Regulated Secretion in Cytotoxic T Lymphocytes
Stinchcombe, Jane C.
Barral, Duarte C.
Mules, Emilie H.
Booth, Sarah
Hume, Alistair N.
Machesky, Laura M.
Seabra, Miguel C.
Griffiths, Gillian M.
The Journal of Cell Biology
Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.
Rab27a; cytotoxic T lymphocyte; secretory lysosomes; immunological synapse; Arp2/3
Related Articles
Statins Inhibit HIV-1 Infection by Down-regulating Rho Activity
del Real, Gustavo
Jiménez-Baranda, Sonia
Mira, Emilia
Lacalle, Rosa Ana
Lucas, Pilar
Gómez-Moutón, Concepción
Alegret, Marta
Peña, Jose María
Rodríguez-Zapata, Manuel
Alvarez-Mon, Melchor
Martínez-A., Carlos
Mañes, Santos
The Journal of Experimental Medicine
Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft–associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We show that statins decreased viral load and increased CD4+ cell counts in acute infection models and in chronically HIV-1–infected patients. Viral entry and exit was reduced in statin-treated cells, and inhibition was blocked by the addition of l-mevalonate or of geranylgeranylpyrophosphate, but not by cholesterol. Cell treatment with a geranylgeranyl transferase inhibitor, but not a farnesyl transferase inhibitor, specifically inhibited entry of HIV-1–pseudotyped viruses. Statins blocked Rho-A activation induced by HIV-1 binding to target cells, and expression of the dominant negative mutant RhoN19 inhibited HIV-1 envelope fusion with target cell membranes, reducing cell infection rates. We suggest that statins have direct anti–HIV-1 effects by targeting Rho.
cholesterol; actin cytoskeleton; small GTPases; lipid rafts; prenylation
Related Articles
How prenylation and S-acylation regulate subcellular targeting and function of ROP GTPases
Sorek, Nadav
Henis, Yoav I
Yalovsky, Shaul
Plant Signaling & Behavior
Rho of Plants (ROP) small G proteins function at discrete domains of the plasma and possibly endo membranes. ROPs are synthesized as soluble proteins and their attachment to membranes and partitioning in membrane microdomains are facilitated by the posttranslational lipid modifications prenylation and/or S-acylation. Based on their amino acid sequences, ROPs can be classified into two major subgroups: type-I ROPs terminate with a canonical CaaX box motif and are prenylated primarily by geranyl-geranyltransferase-I (GGT-I) and to a lesser extent by farnesyltransferase (FT). Type-II ROPs terminate with a plant specific GC-CG box domain and are attached to the plasma membrane by stable S-acylation. In addition, type-I and possibly also type-II ROPs undergo activation dependent transient S-acylation in the G-domain and consequent partitioning into lipid rafts. Surprisingly, although geranylgeranylation is required for the membrane attachment of type-I ROPs and the γ subunits of heterotrimeric G proteins, Arabidopsis mutants lacking GGT-I function have a mild phenotype compared to wild type plants. The mild phenotype of the ggt-I mutants suggested that farnesylation by FT may compensate for the loss of GGT-I function and that possibly the prenylated type-I and S-acylated type-II ROPS have some overlapping functions. In a paper recently published in Plant Physiology1 we examined the role of the prenyl group type in type-I ROP function and membrane interaction dynamics and the functional redundancy between type-I and type-II ROPs. This study complements a second paper in which we examined the role of G-domain transient S-acylation in the membrane interaction dynamics and signaling by type-I ROPs.2 Together these two studies provide a framework for realizing the role of prenylation and S-acylation in subcellular targeting, membrane interaction dynamics and signaling by ROP GTPases.
prenylation; S-acylation; ROP; FRAP; membrane targeting; lipid rafts
Related Articles
Structural Insights into a Unique Legionella pneumophila Effector LidA Recognizing Both GDP and GTP Bound Rab1 in Their Active State
Cheng, Wei
Yin, Kun
Lu, Defen
Li, Bingqing
Zhu, Deyu
Chen, Yuzhen
Zhang, Hao
Xu, Sujuan
Chai, Jijie
Gu, Lichuan
PLoS Pathogens
The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.
Author Summary
Legionella pneumophila delivers 275 validated substrates into the host cytosol by its Dot/Icm type IV secretion system. Several substrates including SidM/DrrA and LidA directly interact with the host Rab GTPases and interfere with the vesicle secretion pathway. SidM/DrrA is necessary for Rab1 recruitment, function as a Rab1 specific GDI displacement factor and guanine nucleotide exchange factor. LidA has the auxiliary activity for Rab1 recruitment, whereas it is more important for the formation of the replication vacuole compared with SidM/DrrA. LidA is predicted to be the first substrate secreted by the Dot/Icm system and is critical for maintaining the integrity of the bacterial cell. Moreover, it expresses throughout the intracellular growth phase, localizes to early secretory compartments, and interacts with several members of Rab family. Here we present the crystal structures of LidA coiled-coil domain in complex with two different states of Rab1, GDP- and GTP-bound. The GDP-bound Rab1 in the complex surprisingly has the same conformation with the GTP-bound Rab1, revealing that LidA can retain Rab1 persistently in its active state. Our structures add a new insight into the regulation of the host Rab1 membrane cycle by pathogen-secreted coiled-coil effector.
Related Articles
Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic β cells
Arora, Daleep K.
Syed, Ismail
Machhadieh, Baker
McKenna, Charles E.
Kowluru, Anjaneyulu
A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet β-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and β-subunits of RGGT in clonal INS 832/13 β-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or β-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.
Geranylgeranylation; Rab G-proteins; Rab escort proteins; insulin secretion; pancreatic β-cells
Related Articles
Measurement of protein farnesylation and geranylgeranylation in vitro, in cultured cells and in biopsies, and the effects of prenyl transferase inhibitors
Berndt, Norbert
Sebti, Saïd M
Nature protocols
The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [3H]farnesyl diphosphate and [3H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [3H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 day (enzyme assays) to up to 3 months (autoradiography of [3H]-labeled proteins).
Related Articles
Driven to Death: Inhibition of Farnesylation Increases Ras Activity in Osteosarcoma and Promotes Growth Arrest and Cell Death
Geryk-Hall, Mandy
Yang, Yanwen
Hughes, Dennis P.M.
Molecular cancer therapeutics
To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibiting malignant growth or inducing apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyl transferase (FTI), the enzyme most often responsible for prenylating Ras, showed cinical promise, but development of FTI such as tipifarnib has been stalled by uncertainty about their mechanism of action, since Ras appeared unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Initial observations of varied response by osteosarcoma cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using osteosarcoma models.
We describe our novel, counter-intuitive finding that endogenous Ras activity increases in osteosarcoma when farnesyl transferase is inhibited either by tipifarnib or shRNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated ERK and p38 MAPK. Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Since Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interact differently with downstream effector proteins in sensitive osteosarcoma cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition.
Osteosarcoma; Ras; farnesylation; Growth arrest
Related Articles
Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway
Sánchez-Mir, Laura
Franco, Alejandro
Martín-García, Rebeca
Madrid, Marisa
Vicente-Soler, Jero
Soto, Teresa
Gacto, Mariano
Pérez, Pilar
Cansado, José
Molecular and Cellular Biology
The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade.
Related Articles
Hsp90 Co-localizes with Rab-GDI-1 and Regulates Agonist-induced Amylase Release in AR42J Cells
Raffaniello, Robert
Fedorova, Daria
Ip, Dawn
Rafiq, Sarwish
Cellular Physiology and Biochemistry
Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI: Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.
Rab proteins; GDI; Hsp90; Ar42J cells; Exocrine; Secretion; Pancreas; Amylase
Related Articles
Alkynyl-farnesol reporters for detection of protein S-prenylation in cells
Charron, Guillaume
Tsou, Lun K.
Maguire, William
Yount, Jacob S.
Hang, Howard C.
Molecular bioSystems
Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.
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Telephone & Addresses
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this Old House
65233 Boonville MO
This House, 49, Albuquerque, S…ha Dr Ne
View This's social profiles and photos on Facebook, MySpace, and +40 Networks.
This House, San Diego, Guizot St
View This's social profiles and photos on Facebook, MySpace, and +40 Networks.
This N House, Broken Arrow, S Beech Ave
View This's social profiles and photos on Facebook, MySpace, and +40 Networks.
Yellow Page contacts
Network Profiles
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Twitter Profile: THIS HOUSE (this_house_)
Location: atlanta, ga / Atlanta Music Guide's ''Ones To Watch'', THIS HOUSE is your new fav indie rock band. @apcurtis89 @camkolar @oparnica
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fotocommunity: I'm gonna quit my job - burn down this house and live out my
2 Pictures with 1 Comment, since 09/2007
Michael Reynolds Beer Can Thumb House - Business Insider
His first creation led to a global enterprise.
David Montelongo AFTER Flip This House - Montelongo Organization |...
David Montelongo AFTER Flip This House. August 10, 2010 -- Fans of Flip This House & David Montelongo may be interested in learning more about what he has been...
Private Homepages
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Bless This House - Welcome to Bless This House.net
A tribute to Bless This House starring Sid James Diana Coupland Sally Geeson and Robin Stewart made by Thames Television,Fremantle Media.Feature film ...
this house is for sale Your Local Estate Agent in Caerphilly,...
this house is for sale your local estate agent Caerphilly, Blackwood, Ystrad Mynach, Bargoed & Cardiff
Adorn This House
The use of the arts in worship is not a luxury. Unless we use our creative gifts to the fullest, our praises to God the Creator will fall flat.
THIS HOUSE Atlanta Music Guide's Ones To Watch, THIS HOUSE is your new favorite indie rock band. THIS HOUSE is Guilmark Bruwer on Drums Austin Curtis on...
Celebrities & Politicians
(1 - 2 from 4
IMDB Filmography: Wenn Mauern töten (TV Movie 1981) - IMDb
Directed by William Wiard. With Parker Stevenson, Lisa Eilbacher, Joan Bennett, Slim Pickens. After having a nervous breakdown, a rock singer has to spend...
IMDB Filmography: Bless This House (TV Series 1995– ) - IMDb
Created by Bruce Helford. With Andrew Dice Clay, Cathy Moriarty, Sam Gifaldi, Raegan Kotz. A lazy and sarcastic postal worker and his wife raise two kids in...
Meghan Carys latest CD: Building This House | Indiegogo
Let's Go Global!!
Oracle Productions – Free Art For All » This House Believes
Oracle Theatre presents. This House Believes the American Dream is at the Expense of the American Negro. Adapted and Directed by Zachary Baker-Salmon.
Books & Literature
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This House of Sky: Landscapes of a Western Mind by Ivan Doig —...
This House of Sky has 3,410 ratings and 366 reviews. Chrissie said: Have your read the novels of Ivan Doig – those, such as The Whistling Season and/or D...
This House Has Fallen: Nigeria in Crisis - Karl Maier - Google Books
To understand Africa, one must understand Nigeria, and few Americans understand Nigeria better than Karl Maier. This House Has Fallen is a bracing and...
This House We Dedicate to Thee
This page contains an interactive player for hymns and children's songs
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Perry Como - Bless This House Lyrics | MetroLyrics
Lyrics to 'Bless This House' by Perry Como. Bless this house, O Lord we pray, / Make it safe by night and day . . . / Bless these walls so firm and stout, /
25/11/2013 · "This House" Lyrics by Gary Barlow: This house, built from stone Bricks and mortar Love and hope (Verse) Voices, children cry Parents teach us...
Gary Barlow - This House Lyrics. (Verse) This house, built from stone Bricks and mortar Love and hope (Verse) Voices, children cry Parents teach us Wrong from right
Here In This House Lyrics - Vicki Yohe
Lyrics to Here In This House by Vicki Yohe: We've gathered here, in faith we believe / We lift our voices, His praises we sing / Whatever
Bless This House (Brahe, May H.) - IMSLP/Petrucci Music Library: Free...
This work likely not be in the public domain in the US (due to first publication with the required notice after 1922, plus renewal or "restoration" ...
Video & Audio
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Brandy - Paint This House - YouTube
More New Music & Videos: http://facebook.com/PlaceForMusic http://facebook.com/PlaceForMusic http://facebook.com/PlaceForMusic Brandy - Paint This House Bran...
Christmas Lights Gone Wild - YouTube
Wow, this thing has really gone crazy with the viewers. Let me first start by saying that this is not my house. Regardless of what any of the comments read, ...
Tracie Spencer - This House - YouTube
Music video by Tracie Spencer performing This House.
Flip This House Full Episodes, Video & More | A&E
Flip This House follows real-estate investors who buy, refurbish, and re-sell properties for a profit.
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Review: 'Welcome to This House,' the …
25.05.2015 · "Welcome to This House," Barbara Hammer's impressionistic yet informative portrait of the poet Elizabeth Bishop, certainly pays tribute to ...
Bless This House - TV.com
Bless This House: Watch full length episodes & video clips. Read the latest Bless This House episode guides & recaps, fan reviews, news, and much more.
Flip This House - TV.com
Flip This House: Watch full length episodes & video clips. Read the latest Flip This House episode guides & recaps, fan reviews, news, and much more.
Sell This House - TV.com
Sell This House: Watch full length episodes & video clips. Read the latest Sell This House episode guides & recaps, fan reviews, news, and much more.
Reports & Statements
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Wikipedia: Bless This House (song) - Wikipedia, the free encyclopedia
"Bless This House" is a song published in 1927. The words were written by Englishwoman Helen Taylor, under the original title "Bless the House."
Wikipedia: Sell This House - Wikipedia, the free encyclopedia
Sell This House is a reality television series that started airing on the A&E television network in 2003. Host Tanya Memme and designer/home stager Roger ...
Wikipedia: Bless This House (film) - Wikipedia, the free encyclopedia
Bless This House is a 1972 British comedy film and a spin off from the television sitcom Bless This House. Contents 1 Plot 2 Cast 3 Filming locations 4 External links ...
Wikipedia: Bless This House - Wikipedia, the free encyclopedia
Bless This House may refer to: Bless This House (British TV series) a British sitcom. Bless This House (film) a film spin-off of the above Bless This House (U.S. TV ...
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13th Floor Elevators Slip Inside This House Lyrics | Lyrics007
Read guaranteed accurate human-edited 13th Floor Elevators Slip Inside This House lyrics from lyrics007
5,000 Venomous Spiders Took Over This House In The Spirit Of...
1 hour ago. 5,000 Venomous Spiders Took Over This House In The Spirit Of Halloween And It's Terrifying. James B. Barnes. View Comments. Content reported.
Bless This House DVD Set - £33.97 : Classic Movies on DVD from...
Classic Movies on DVD Bless This House DVD Set - Bless this House dvd set, containing the complete series 1-6 on 12 dvd's. DELIVERED FREE IN THE UK. The...
Can You Escape This House 2 Walkthrough - FreeAppGG
Can You Escape This House 2 Walkthrough
Bless This House, Hymnlyrics.org
Bless this house, Oh Lord we pray. Make it safe by night and day. Bless these walls so firm and stout. Keeping want and trouble out. Bless the roof and chimneys ...
Step Inside This House - Lyle Lovett | Songs, Reviews, Credits |...
Find album reviews, stream songs, credits and award information for Step Inside This House - Lyle Lovett on AllMusic - 1998 - Step Inside This House, in a way,...
Can You Escape This House Level 8 Walkthrough - FreeAppGG
Can You Escape This House Level 8 Walkthrough
THIS HOUSE | Free Listening on SoundCloud
Guilmark Bruwer - Drums Austin Curtis - Guitar Cameron Kolar - Vocals and Guitar Jef Oparnica - Bass. acworth, ga. 5 Tracks. 75 Followers. Stream Tracks and...
Isaac Carree Feat. R. Kelly "Clean This House" (Remix) [VIDEO] | The...
Isaac Carree and R.Kelly duet with a plead to restore their souls in this
It's too many hoes in this house of sin – On Sight Lyrics Meaning
Find out the meaning behind this lyric from On Sight by Kanye West. Get more than just the lyrics on Genius
In This House On This Morning – Wynton Marsalis Official Website
The audience caught fire when the Wynton Marsalis Septet premiered this full scale suite, at a gala concert in Lincoln Center's Avery Fisher Hall on May 27,...
In This House, On This Morning - Wynton Marsalis | Songs, Reviews,...
Find album reviews, stream songs, credits and award information for In This House, On This Morning - Wynton Marsalis on AllMusic - 1993 - For this double CD,...
This House - National Theatre Live
Biting and energetic, This House has enjoyed two National Theatre sell-out runs. Read full synopsis. This production is no longer showing. What's on now ...
Star of 'Flip This House' sues brother - San Antonio Express-News
The San Antonio-based businessman, who gained fame in 2006 as a star of the A&E show "Flip This House," is suing brother and former co-star David Montelongo...
This House | National Theatre | South Bank, London
Following a sell-out run in the Cottesloe, James Graham's acclaimed play transfers to the Olivier Theatre from February 2013.
THIS HOUSE by Daniel Hymanson —Kickstarter
Daniel Hymanson is raising funds for THIS HOUSE on Kickstarter! a collaborative film project about a dreamy place, memories of childhood, and a union of...
Beliebte Artikel für in this house we do auf Etsy
Meide Kaufhäuser und Handelsketten – kaufe einzigartige Artikel für in this house we do bei Tausenden von unabhängigen Designern und Vintage-Sammlern auf Etsy...
This House is Haunted by Brain Punch Games —Kickstarter
Brain Punch Games is raising funds for This House is Haunted on Kickstarter! Your house is haunted by demonic spirits! Will you banish the evil presence or...
"This House" — Scriggler
While walking to church one day, I was struck by this house. I could not stop thinking about it, all through the service. Later that day it prompted me to write this ...
ALISON MOYET LYRICS - This House - A-Z Lyrics
Lyrics to "This House" song by ALISON MOYET: Whose sticky hands are these And what is this empty place I could be happily lost But for your face...
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