Biology subjects

Pasupulati, A. k.

Publications and source records attributed to Pasupulati, A. k..

6 recordsLinked to original sources

bioRxiv2026

CD2AP's structure and oligomerization are compromised by the K301M mutation: implications for Nephrotic syndrome

IntroductionThe podocyte slit diaphragm (SD) is a complex filtration unit localized to the blood and urine interface and governs the glomerular selectivity. However, the greater details of the SD composition and the mechanism of assembly of the SD protein as a macromolecular complex remain elusive. CD2-associated protein (CD2AP) serves as a central scaffold within the SD, and mutations in CD2AP are strongly associated with nephrotic syndrome (NS) and focal segmental glomerulosclerosis (FSGS). However, the mechanisms by which such mutations alter the architecture and higher-order organization of CD2AP are poorly understood. MethodsWe employed biophysical, structural, and proteomic approaches to investigate the impact of the disease-associated K301M mutation on CD2AP structure and its interaction with Podocin. Oligomerization was analyzed using size-exclusion chromatography, blue native PAGE, Dynamic light scattering, and small-angle X-ray scattering. Secondary and tertiary structural properties were assessed by far- and near-UV circular dichroism, thermal denaturation, and intrinsic fluorescence spectroscopy. CD2AP-podocin interactions were quantified using in vitro pulldown and surface plasmon resonance (SPR), and mutation-dependent changes in interaction networks were examined through interactome profiling. ResultsWild-type (WT) CD2AP assembled into flexible higher-order oligomers ([~]9-12-mers), whereas the K301M variant collapsed into lower-order species ([~]3-6-mers), indicating destabilization of the coiled-coil assembly interface. Spectroscopic analyses revealed subtle secondary-structure rearrangements, but profound tertiary packing defects, as well as reduced and markedly diminished thermal resilience in the mutant. SPR analysis demonstrated loss of binding between Podocin and mutant CD2AP, whereas WT CD2AP showed high-affinity interaction (KD = 211 nM) with Podocin. Complementary interactome profiling revealed widespread rewiring of protein-protein interactions in the case of mutant CD2AP, characterized by the loss of core partners and the emergence of aberrant associations. ConclusionThese findings define a mechanistic model in which the K301M mutation destabilizes CD2AP oligomerization, disrupts podocin recognition, and remodels interaction networks essential for SD stability. This work signifies the importance of CD2AP in SD assembly and the permselective filtration function of the kidney, and the impact of a single mutation in the pathogenesis of NS and FSGS. Translational Statement: Inherited nephrotic syndrome, characterized by heavy proteinuria, frequently arises from mutations in scaffolding proteins of the slit-diaphragm (SD). This study demonstrates that the nephrotic syndrome-associated K301M mutation in CD2-associated protein (CD2AP) compromises higher-order oligomerization, abolishes its binding to the binding partner (Podocin), and reshapes protein-protein interaction networks that are critical for SD assembly and stability. By establishing a direct link between mutation-induced collapse of CD2AP architecture and loss of podocyte scaffolding function, these findings provide mechanistic insight into the pathogenesis of CD2AP-associated proteinuric kidney disease. The results further identify oligomeric assembly interfaces as potential targets for therapeutic strategies aimed at preserving the integrity of the SD and glomerular filtration function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/698362v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1f49f1dorg.highwire.dtl.DTLVardef@fa2e06org.highwire.dtl.DTLVardef@e399c0org.highwire.dtl.DTLVardef@831c17_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biophysics↗

bioRxiv2025

Structural conformations of intrinsically disordered proteins of podocyte slit-diaphragm

The slit-diaphragm (SD), a specialized junction between the foot processes of neighboring podocytes, regulates the permselectivity of glomerular filtration. It functions as a size- and charge-selective molecular sieve, ensuing protein-free urine. SD comprises several proteins such as nephrin, NEPH1, podocin, CD2AP, and TRPC6. Nephrin and NEPH1 are extracellular proteins that bridge the gap between foot processes, whereas podocin and CD2AP are adapter proteins. The intrinsically disordered regions (IDRs) of SD proteins play a crucial role in assembling these proteins as a macromolecular complex. Mutations in these proteins disrupt SD integrity, leading to a nephrotic syndrome characterized by heavy proteinuria. The structural details of each protein of this macromolecular complex are poorly detailed. We employed molecular docking and molecular dynamics simulations to investigate the structural dynamics of SD proteins. Our findings reveal that SD proteins exhibit partner-specific conformational adaptations driven by short linear motifs and molecular recognition features. CD2AP interacts transiently with nephrin but forms a more stable complex with podocin. NEPH1 and nephrin interact via extracellular immunoglobulin domains while maintaining dynamic intracellular contacts. Podocin preferentially interacts with nephrin over NEPH1, and distinct subunit-sharing mechanisms emerge, where CD2AP and TRPC6 may simultaneously associate with a single podocin subunit. Mutational analysis reveals that disease-associated variants, including CD2AP (P532S), podocin (R138Q), and nephrin (G1161V), enhance local stability but restrict the flexibility of IDR and impair SD assembly. Our study provides evidence of dynamic subunit sharing within the SD complex, offering new insights into the assembly of SD in health and disease.

bioinformatics↗

bioRxiv2021

ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

Glomerular podocytes are instrumental in ensuring glomerular permselectivity and regulating the integrity of glomerular biology. However, podocytes are vulnerable to various noxious stimuli such as hypoxia, and podocyte injury presented with glomerulosclerosis and impaired kidney function. The mechanism of hypoxia-induced podocyte injury vis-a-vis glomerulosclerosis has remained enigmatic. Hypoxia inducible factor 1 (HIF1) that transduces hypoxic adaptations, induces Transglutaminase 2 (TG2), a calcium dependent enzyme that catalyzes intramolecular {varepsilon}-({gamma}-glutamyl) lysine cross-links of extracellular matrix (ECM) proteins. In this study, we investigated the mechanism of regulation of TG2 by HIF1. Stabilization of HIF1 by FG4592 (Roxadustat) and physiological hypoxia, resulted in elevated expression of ZEB2 (zinc-finger E-box-homeobox 2) and its downstream target TRPC6 (transient receptor potential channel 6). ZEB2 transcriptionally activates TG2 expression, whereas, via TRPC6, it induces calcium influx, inturn it increases the TG2 activity. Blocking the TRPC6 action or suppressing its expression only partially attenuated FG4592 induced TG2 activity, whereas suppression of ZEB2 expression significantly abolished TG2 activity. This study demonstrates that stabilization of HIF1 stimulates both TG2 expression and activity, whereas abrogation of HIF1 by metformin prevented HIF1 regulated TG2 and consequent glomerular injury.

cell biology↗

bioRxiv2020

Molecular Evolution of Podocyte Slit-diaphragm Proteins

Vertebrates kidneys contribute to the homeostasis by regulating electrolyte, acid-base balance, and prevent protein loss into the urine. Glomerular podocytes constitute blood-urine barrier and podocyte slit-diaphragm, a modified tight junction contributes to the glomerular permselectivity. Nephrin, podocin, CD2AP, and TRPC6 are considered to be crucial members, which largely interact with each other and contribute to the structural and functional integrity of the slit-diaphragm. In this study, we analyzed the distribution of these four-key slit-diaphragm proteins across the organisms for which the genome sequence is available. We found that nephrin has a diverse distribution ranging from nematodes to higher vertebrates whereas podocin, CD2AP, and TRPC6 are predominantly restricted to the vertebrates. In the invertebrates nephrin and its orthologs consist of more immunoglobulin-3 and immunoglobulin-5 domains when compared to the vertebrates wherein, CD80-like C2-set Ig2 domains were predominant. Src Homology-3 (SH3) domain of CD2AP and SPFH domain of podocin are highly conserved among vertebrates. Although the majority of the TRPC6 and its orthologs had conserved ankyrin repeats, TRP, and ion transport domains, the orthologs of TRPC6 present in Rhincodon typus and Acanthaster planci do not possess the ankyrin repeats. Intrinsically unstructured regions (IURs), which are considered to contribute to the interactions among these proteins are largely conserved among orthologs of these proteins, suggesting the importance of IURs in the protein complexes that constitute slit-diaphragm. This study for the first time reports the evolutionary insights of vertebrate slit-diaphragm proteins and its invertebrate orthologs.

evolutionary biology↗

bioRxiv2020

Structural Features and Oligomeric Nature of Human Podocin Domain

Podocytes are crucial cells of the glomerular filtration unit and playing a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and shape-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101-125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126-350). We show that the podocin domain majorly homo-oligomerize into a 16mer. Circular dichroism and fluorescence spectroscopy suggest that the 16mer oligomer has considerable secondary structure and moderate tertiary packing.

biophysics↗

bioRxiv2019

Growth hormone induces TGF-β1 in podocytes: Implications in podocytopathy and proteinuria

Podocytes are integral members of the filtration barrier in the kidney and are crucial for glomerular permselectivity. Podocytes are highly differentiated and vulnerable to an array of noxious stimuli during various clinical conditions whereas podocyte loss plays a key role in progressive glomerular diseases. Elevated circulating growth hormone (GH) levels are associated with podocyte injury and proteinuria in diabetics. Previous studies have shown that podocytes express GH receptors (GHR), and induce Notch signaling when exposed to GH. However, the precise mechanism(s) by which excess GH elicits podocytopathy remains to be elucidated. In the present study, we demonstrate that GH induces cognate TGF-{beta}1 signaling and provokes cell cycle re-entry of otherwise quiescent podocytes. Though, differentiated podocytes re-enter the cell cycle in response to GH and TGF-{beta}1 unable to accomplish cytokinesis, despite nuclear division. Owing to this aberrant cell-cycle events significant amount of GH or TGF-{beta}1 treated cells remain binucleated and undergo mitotic catastrophe. Importantly, inhibition of GHR, TGFBR1, or Notch signaling prevented cell cycle re-entry and protects podocyte from cell death. Furthermore, inhibition of Notch activation prevents GH-dependent podocyte injury and proteinuria. Kidney biopsy sections from patients with diabetic nephropathy show activation of Notch signaling and bi-nucleated podocytes. All these data confirm that excess GH induces Notch1 signaling via TGF-{beta}1 and contributes to the mitotic catastrophe of podocytes. This study highlights the role of aberrant GH signaling in the podocytopathy and the potential application of inhibitors of TGF-{beta}1 or Notch inhibitors as a therapeutic agent for diabetic nephropathy. Significance StatementElevated circulating levels of growth hormone (GH) associated with glomerular hypertrophy and proteinuria. Whereas decreased GH action protected against proteinuria. Podocytes are highly differentiated cells that play a vital role in glomerular filtration and curb protein loss. The direct role of GH in podocytes is the focus of our study. We found that GH induces TGF-{beta}1 and both provoke cell cycle re-entry of podocytes in Notch1 dependent manner. Notch activation enables the podocytes to accomplish karyokinesis, but not cytokinesis owing to which podocytes remain binucleated. Binucleated podocytes that were observed during GH/TGF-{beta}1 treatment are susceptible to cell death. Our study highlighted the fact that enforcing the differentiated podocytes to re-enter the cell cycle results in mitotic catastrophe and permanent loss.

cell biology↗