斯蒂芬·威尔肯斯博士

研究抽象

Our main focus is on the structure and mechanism of the eukaryotic proton pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase). v - atp酶是一个很大的, multisubunit membrane protein complex found on the endomembrane system of all eukaryotic cells, where it functions to acidify intracellular organelles, 包括核内体, 溶酶体, the Golgi apparatus and synaptic vesicles. An acidic pH in the lumen of these compartments is essential for basic cellular functions including endocytosis, 蛋白质的降解, 蛋白质贩卖, and neurotransmitter loading. When found on the plasma membrane of "professional" acid secreting cells such as bone osteoclasts and renal intercalated cells, V-ATPase pumps protons to the outside of the cell, a process required for bone remodeling and urine acidification. Due to its involvement in numerous basic cellular functions, a malfunctioning V-ATPase can lead to widespread human diseases including osteoporosis, 神经退化, 糖尿病, 癌症, 和艾滋病.

V-ATPase is composed of two rotary motor subcomplexes, a membrane extrinsic ATP hydrolyzing V1-ATPase, and a membrane embedded Vo proton turbine. ATP hydrolysis on V1 is coupled to proton transport across Vo by a "central stalk" that rotates up to 100 1/s to pick up protons from the cytosol and deliver them to the organelle lumen or outside of the cell. As a major consumer of cellular energy, V-ATPase function is tightly regulated by a mechanism referred to as "reversible disassembly". When the enzyme's proton pumping is not needed, V1 dissociates from the membrane bound Vo, and the activity of both subcomplexes is "silenced" so that V1 is no longer capable of hydrolyzing MgATP, and Vo becomes impermeable to protons. Reversible disassembly is unique to eukaryotic V-ATPase and the process offers a way to modulate the activity of a disease causing V-ATPase for therapeutic purposes.

We work with the V-ATPases from the model organism Saccharomyces cerevisiae (baker's yeast) as well as human. We are using the tools of biochemistry, molecular & 细胞生物学, biophysics and structural biology (including NMR spectroscopy, X-ray crystallography and cryo electron microscopy (cryoEM)) to obtain structural and mechanistic information. Our long term goal is to elucidate the mechanism of V-ATPase's unique mode of regulation and find ways for therapeutic targeting of the enzyme in the disease state.

最近的出版物

沙玛,年代.， t, R.A.m.m. 威尔肯斯，S. (2019) Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of regulation by reversible disassembly. J. 医学杂志. 化学.，出版中.

沙玛,年代.， t, R.A. 威尔肯斯，S. (2018) MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly. J. 医学杂志. 化学. 293, 10718-10730.

卢武铉,年代.-H.，斯坦，N.J.C.库恩-克德尔，S.， Pintillie, G, Chiu, W. 威尔肯斯，S. (2018).5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel. 摩尔. 细胞 69, 993-1004.

Oot R.A.库-卡德尔，S.夏尔马，S.，斯坦，N.J. 威尔肯斯，S. (2017) Breaking up and Making up: The Secret Life of the Vacuolar H+-ATPase. 蛋白质科学 26, 896-909.

沙玛,年代. 威尔肯斯，S. (2017) 医学杂志ayer Interferometry of Lipid Nanodisc-Reconstituted Yeast Vacuolar H+-ATPase. 蛋白质科学 26, 1070-1079.

斯塔姆,N.J. 威尔肯斯，S. (2017) Structure of Nanodisc Reconstituted Vacuolar ATPase Proton Channel: Definition of the Interaction of Rotor and Stator and Implications for Enzyme Regulation by Reversible Dissociation. J. 医学杂志. 化学. 292, 1749-61.

Oot R. A.凯恩，P.M.贝瑞.A. 威尔肯斯，S. (2016) Crystal Structure of Yeast V1-ATPase in the Autoinhibited State. EMBO J. 35,1694-706.

Couoh-Cardel,年代.， eh, Y.-C.威尔肯斯，S. 和莫维利亚努，L. (2016) Yeast V-ATPase Proteolipid Ring Acts as a Large-Conductance Transmembrane Protein Pore. Sci. 代表. 6, 24774.