VHL-P138R and VHL-L163R Novel Variants: Mechanisms of VHL Pathogenicity Involving HIF-Dependent and HIF-Independent Actions.

The von Hippel-Lindau (VHL) disease is an autosomal dominant cancer syndrome caused by mutations in the VHL tumor suppressor gene. VHL protein (pVHL) forms a complex (VBC) with Elongins B-C, Cullin2, and Rbx1. Although other functions have been discovered, the most described function of pVHL is to recognize and target hypoxia-inducible factor (HIF) for degradation. This work comprises the functional characterization of two novel variants of the VHL gene (P138R and L163R) that have been described in our center in patients with VHL disease by in vitro, in vivo, and in silico approaches. In vitro, we found that these variants have a significantly shorter half-life compared to wild-type VHL but still form a functional VBC complex. Altered fibronectin deposition was evidenced for both variants using immunofluorescence. In vivo studies revealed that both variants failed to suppress tumor growth. By means of molecular dynamics simulations, we inspected in silico the nature of the changes introduced by each variant in the VBC complex. We have demonstrated the pathogenicity of P138R and L163R novel variants, involving HIF-dependent and HIF-independent mechanisms. These results provide the basis for future studies regarding the impact of structural alterations on posttranslational modifications that drive pVHL's fate and functions.

Understanding the mechanism of H2S oxidation by flavin-dependent sulfide oxidases: a DFT/IEF-PCM study.

In the last years, H2S has been recognized as a signaling molecule in mammals, which can synthesize and catabolize (by oxidation) such species. The latter process is accelerated by a sulfide:quinone oxidoreductase (SQR, E.C. 1.8.5.4), a flavin-dependent sulfide oxidase (FDSO). FDSOs catalyze electron transfer from H2S to an acceptor in catalytic cycles involving two phases: (I) reduction of FAD by H2S (SH-) and (II) electron transfer from FADH- to the electron acceptor. The first step of FAD reduction consists on the reaction of SH- with a catalytic disulfide at the active site of the enzyme, to yield a thiolate and a persulfide in the protein. This step is ca. 106 times faster than the analogous reaction with low-molecular-weight disulfides (LMWDs) and the causes of such extraordinary acceleration remain unknown. Using the IEF-PCM(ε ≈ 10)/M06-2X-D3/6-31+G(d,p) level of theory, we have modeled the reaction of SH- with a disulfide as located in a representative model of the active site extracted from a prokaryotic SQR, assessing the effects of partial covalent interactions (PCIs) between the leaving sulfur atom and flavin ring on the activation Gibbs free-energy barrier at 298 K (∆‡G298K). To also evaluate the importance of entropic penalties on the first step, we have modeled at the same level of theory the reaction of (bis)hydroxyethyl disulfide in aqueous solution, a LMWD for which experimental data is available. Our results show that PCIs between the leaving sulfur atom and the flavin group only have a minor effect (∆‡G298K reduced by 1.6 kcal mol-1) while compensating entropic penalties could have a much larger effect (up to 8.3 kcal mol-1). Finally, we also present here a first model of some of further steps in the phase I of the catalytic cycle as in mammalian FDSOs, providing some light about their detailed mechanism. Graphical abstract .

Reaction of Hydrogen Sulfide with Disulfide and Sulfenic Acid to Form the Strongly Nucleophilic Persulfide.

Hydrogen sulfide (H2S) is increasingly recognized to modulate physiological processes in mammals through mechanisms that are currently under scrutiny. H2S is not able to react with reduced thiols (RSH). However, H2S, more precisely HS(-), is able to react with oxidized thiol derivatives. We performed a systematic study of the reactivity of HS(-) toward symmetric low molecular weight disulfides (RSSR) and mixed albumin (HSA) disulfides. Correlations with thiol acidity and computational modeling showed that the reaction occurs through a concerted mechanism. Comparison with analogous reactions of thiolates indicated that the intrinsic reactivity of HS(-) is 1 order of magnitude lower than that of thiolates. In addition, H2S is able to react with sulfenic acids (RSOH). The rate constant of the reaction of H2S with the sulfenic acid formed in HSA was determined. Both reactions of H2S with disulfides and sulfenic acids yield persulfides (RSSH), recently identified post-translational modifications. The formation of this derivative in HSA was determined, and the rate constants of its reactions with a reporter disulfide and with peroxynitrite revealed that persulfides are better nucleophiles than thiols, which is consistent with the α effect. Experiments with cells in culture showed that treatment with hydrogen peroxide enhanced the formation of persulfides. Biological implications are discussed. Our results give light on the mechanisms of persulfide formation and provide quantitative evidence for the high nucleophilicity of these novel derivatives, setting the stage for understanding the contribution of the reactions of H2S with oxidized thiol derivatives to H2S effector processes.