Hydrogen sulfide metabolism in cancer and homocystinuria: towards the development of new pharmacological strategies

Hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), belongs to a small group of gaseous signalling molecules termed ‘gasotransmitters’. H2S is involved in important physiological and pathological processes and exerts a dose-dependent effect on cellular bioenergetics, acting at higher levels as an inhibitor of mitochondrial cytochrome c oxidase or stimulating ATP synthesis at lower concentrations. A growing number of pathologies are reportedly associated with alterations of H2S metabolism. Here, the human H2S metabolism was investigated in classical homocystinuria and in cancer; furthermore, it was explored a new pharmacological strategy against cancer based on human ferritin (Hft) as a drug delivery system. Classical homocystinuria is a rare genetic disease, associated with mutations in the gene encoding the heme-containing enzyme cystathionine β-synthase (CBS), a major source of H2S in humans. CBS is allosterically activated by S-adenosyl-L-methionine, but inhibited by CO or NO. Here, it was found that the pathogenic p.P49L variant of human CBS, as produced in E. coli and purified, displays reduced affinity for the PLP (pyridoxal 5’-phosphate) cofactor and increased affinity of the ferrous heme for CO. This could lead to enzyme inhibition at physiological CO concentrations, thereof representing a pathogenic mechanism in classical homocystinuria. Hydrogen sulfide plays an important role also in colon cancer where it promotes angiogenesis and, interestingly, energy metabolism. Here, the H2S catabolism was explored in colon cancer model cells (SW480) exposed to hypoxia, a common factor of tumoral microenvironment that promotes tumor cell survival and propagation. Cells were grown under either normoxic (20% O2) or hypoxic (1% O2) conditions and their ability to metabolize H2S at the mitochondrial level was assayed by high resolution respirometry. Intriguingly, exposure to hypoxic conditions for 24 hours, while reducing the mitochondrial mass and the maximal efficacy to metabolize sulfide, was found to increase the expression of the H2S-metabolizing mitochondrial enzyme sulfide:quinone oxidoreductase. These effects induced by hypoxia may affect the H2S levels in SW480 cells, pointing to H2S catabolism as a potential drug target in colon cancer. Finally, in order to design new therapeutic strategies against cancer, it was obtained an engineered human ferritin-based nanocarrier (HFt-MP-PASE), able to effectively incorporate and deliver the chemotherapeutic drug mitoxantrone with high killing efficacy against model cells of colon cancer progression (SW480 and SW620)

Hydrogen sulfide oxidation: Adaptive changes in mitochondria of SW480 colorectal cancer cells upon exposure to hypoxia

Hydrogen sulfide (H2S), a known inhibitor of cytochrome c oxidase (CcOX), plays a key signaling role in human (patho)physiology. H2S is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide:quinone oxidoreductase (SQR), whereby H2S-derived electrons are injected into the respiratory chain stimulating O2 consumption and ATP synthesis. Under hypoxic conditions, H2S has higher stability and is synthesized at higher levels with protective effects for the cell. Herein, working on SW480 colon cancer cells, we evaluated the effect of hypoxia on the ability of cells to metabolize H2S. The sulfide-oxidizing activity was assessed by high-resolution respirometry, measuring the stimulatory effect of sulfide on rotenone-inhibited cell respiration in the absence or presence of antimycin A. Compared to cells grown under normoxic conditions (air O2), cells exposed for 24 h to hypoxia (1% O2) displayed a 1.3-fold reduction in maximal sulfide-oxidizing activity and 2.7-fold lower basal O2 respiration. Based on citrate synthase activity assays, mitochondria of hypoxia-treated cells were 1.8-fold less abundant and displayed 1.4-fold higher maximal sulfide-oxidizing activity and 2.6-fold enrichment in SQR as evaluated by immunoblotting. We speculate that under hypoxic conditions mitochondria undergo these adaptive changes to protect cell respiration from H2S poisoning

The kinetics of folding of the NSH2 domain from p85

SH2 domains are protein domains that mediate protein-protein interaction through the recognition and binding of specific sequences containing phosphorylated tyrosines. The p85 protein is the regulatory subunit of the heterodimeric enzyme PI3K, an important enzyme involved in several molecular pathways. In this work we characterize the folding kinetics of the NSH2 domain of p85. Our data clearly reveal peculiar folding kinetics, characterized by an apparent mismatch between the observed folding and unfolding kinetics. Taking advantage of double mixing stopped flow experiments and site directed mutagenesis we demonstrate that such behavior is due to the cis/trans isomerization of the peptide bond between D73 and P74, being in a cis conformation in the native protein. Our data are discussed in comparison with previous works on the folding of other SH2 domains

N-acetylcysteine serves as substrate of 3-mercaptopyruvate sulfurtransferase and stimulates sulfide metabolism in colon cancer cells

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains

The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure–function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, in-fluencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts

Understanding the Binding Induced Folding of Intrinsically Disordered Proteins by Protein Engineering: Caveats and Pitfalls

Many proteins lack a well-defined three-dimensional structure in isolation. These proteins, typically denoted as intrinsically disordered proteins (IDPs), may display a characteristic disorder-to-order transition when binding their physiological partner(s). From an experimental perspective, it is of great importance to establish the general grounds to understand how such folding processes may be explored. Here we discuss the caveats and the pitfalls arising when applying to IDPs one of the key techniques to characterize the folding of globular proteins, the Φ value analysis. This method is based on measurements of the free energy changes of transition and native states upon conservative, non-disrupting, mutations. On the basis of available data, we reinforce the validity of Φ value analysis in the study of IDPs and suggest future experiments to further validate this powerful experimental method

The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events

Understanding the mechanism of recognition of gab2 by the n-sh2 domain of shp2

Gab2 is a scaffold protein with a crucial role in colocalizing signaling proteins and it is involved in the regulation of several important molecular pathways. SHP2 is a protein phosphatase that binds, through its two SH2 domains, specific consensus sequences presenting a phosphorylated tyrosine located on the disordered tail of Gab2. To shed light on the details of such a fundamental interaction for the physiology of the cell, we present a complete mutational analysis of the kinetics of binding between the N-SH2 domain of SHP2 and a peptide mimicking a specific region of Gab2. By analyzing kinetic data, we determined structural features of the transition state of the N-SH2 domain binding to Gab2, highlighting a remarkable cooperativity of the binding reaction. Furthermore, comparison of these data with ones previously obtained for another SH2 domain suggests the presence of underlying general features characterizing the binding process of SH2 domains. Data are discussed under the light of previous works on SH2 domains

Experimental Characterization of the Interaction between the N-Terminal SH3 Domain of Crkl and C3G

Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains

Folding and misfolding of a PDZ tandem repeat

Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of whirlin (PDZ1 and PDZ2 - namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics