Biology subjects

Wilkens, S.

Publications and source records attributed to Wilkens, S..

4 recordsLinked to original sources

bioRxiv2025

Interaction of yeast V-ATPase with TLDc protein Rtc5p

The eukaryotic vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes, a mode of regulation conserved from yeast to humans. While signals that govern V-ATPase assembly have been studied in the cellular context, the molecular mechanisms of the process at the level of the enzyme remain poorly understood. We recently discovered that Oxr1p, one of the two TLDc domain proteins in yeast, is essential for rapid V-ATPase disassembly in vivo. How the second TLDc protein, Rtc5p, functions in reversible disassembly, however, is less clear. Here we find that Rtc5p promotes assembly of functional holo V-ATPase from purified V1 and Vo subcomplexes in vitro. CryoEM structures show that Rtc5ps TLDc domain binds the C-terminal domain of the V1-B subunit, with Rtc5ps C-terminal a-helix inserting into the catalytic hexamer, thereby opening a second catalytic site distal to its binding site. Unlike Oxr1p, however, which when deleted produces a distinct phenotype, Rtc5p does not appear to be essential for glucose driven enzyme (re)assembly, hinting at the presence of multiple assembly pathways in vivo.

biochemistry↗

bioRxiv2025

Monoclonal Nanobodies Alter the Activity and Assembly of the Yeast Vacuolar H+-ATPase

The vacuolar ATPase (V-ATPase; V1Vo) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase Vo proton channel subcomplex. Here, we present an in-depth characterization of three anti-Vo Nbs using biochemical and biophysical in vitro experiments. We find that the Nbs bind Vo with high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind the c subunit ring of the Vo on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast Vo can pull down human V-ATPase (HsV1Vo). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.

biochemistry↗

bioRxiv2023

Mimicking Kidney Flow Shear Efficiently Induces Aggregation of LECT2, a Protein Involved in Renal Amyloidosis

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Homozygosity of the I40V LECT2 mutation is believed to be necessary but not sufficient for the disease. Previous studies established that LECT2 fibrillogenesis is greatly accelerated by loss of its single bound zinc ion and stirring or shaking. These forms of agitation are often used to facilitate protein aggregation, but they create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress--shear generated by fluid flow through a network of artery and capillary-sized channels--drives LECT2 fibrillogenesis. To mimic blood flow through the human kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 m in width. Flow shear was particularly pronounced at the branch points and in the smallest capillaries, and this induced LECT2 aggregation much more efficiently than conventional shaking methods. EM images suggested the resulting fibril structures were different in the two conditions. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both size and density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering the mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

biochemistry↗

bioRxiv2018

Role of the H subunit C-terminal domain in the assembly of the vacuolar H+-ATPase

The vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor and recent structural analysis revealed that disassembled V1 and Vo are in different rotary states, a mismatch that is likely responsible for the inability to reconstitute holo V-ATPase from its functional sectors in vitro. Here, using the model organism S. cerevisiae, we show that a key impediment for binding of autoinhibited V1 to Vo is the conformation of the inhibitory C-terminus of subunit H (HCT). Using biolayer interferometry and biochemical analysis, we show that selective disruption of HCTs binding site on V1 allows in vitro assembly of a structurally and functionally coupled V-ATPase complex. The resultant mutant V-ATPase, however, does not disassemble as readily as the wild type enzyme, highlighting the importance of HCTs conformation in the mechanism of reversible disassembly. These findings pave the way for identifying molecules that allow for therapeutic modulation of aberrant V-ATPase activity in the disease state.

biochemistry↗