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APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin

Summary The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

(C) Immunofluorescence analysis of APOL1 and APOL3 distribution with respect to ER and trans-Golgi markers (Calnexin and Golgin97, respectively). The graphs show the colocalization levels of cytoplasmic APOLs with ER or trans-Golgi markers, following removal of the APOL staining covered with the nuclear DAPI marker (error bars = SD).
Cells were randomly selected from 3 independent experiments (ctrl = untreated cells; n = nucleus; scale bars = 20 µm). In all images, the nuclear and cellular contours are indicated by dotted lines; when absent, these contours are outside the image surface.  Size bars = 2 µm. All quantitative measurements are expressed as means +/-SD. Size bars = 2 µm. All quantitative measurements are expressed as means +/-SD.     (E) Sequence comparison of the APOL1 and APOL2 SIDs (asterisks = identical amino acids; underlined violet amino acids: hydrophobic residues in HCs; underlined pink amino acids: positions "a" and "d" of heptads in LZs).   Left panels illustrate clustered labelling close to the mitochondrion (m = mitochondrion); panels at the right exhibit cytosolic labelling. Bars = 200 nm.
(B) Western blot analysis of APOL1 in either normal human serum (NHS) (0.2 µl) or WT podocytes extracts (10 µg). NHS was added to extracts from either 1KO or WT podocytes to avoid any electrophoretic mobility difference linked to differential protein loading, also allowing the demonstration that intracellular APOL1 (arrow) migrates between the two APOL1 bands in NHS.
Coomassie blue staining allows detection of NCS-1 (arrowhead), in total E. coli extracts or purified recombinant protein fraction.
(G) BLI measurements of interaction between bound NCS-1 and APOL3, alone or with different molar ratios of G1 or G2 (n = 3).
All quantitative measurements are expressed as means +/-SD.  Whereas APOL1 or APOL1 G1/G2 variants can hinder association of APOL3 with NCS-1 (dotted or plain red line respectively), APOL1∆ can bind to APOL3 and inactivate this protein without dissociating the APOL3/NCS-1 complex. In APOL1∆ and APOL3KO cells where APOL3 is no longer active, PI4KB is inhibited and the level of Golgi PI(4)P is reduced. However, in the absence of both APOL1 and APOL3, PI4KB appears to be active. In addition to the control of PI4KB, APOL1 and APOL3 also influence the levels of Ca 2+ in Golgi stores, possibly through their redundant K + channel activity. Hypotheses attempting to explain these observations are presented in the discussion (CALN = PI4KB inhibitor calneuron).
(B) Interplay between APOLs, PI(4)P and the NM2A myosin. APOL1 and APOL3 bind to and co-localize with PI(4)P, and indirectly associate with the fission and secretion motor NM2A. Multiple pathways connect PI(4)P with NM2A. In particular, (i) the PI(4)Pbinding protein GOLPH3 binds to the NM2A-interacting protein MYO18A (Billington et