Developing proteomics approaches for identifying new, redox-regulated proteins

Abstract only availableFor many years it was thought that hydrogen peroxide was only a toxic substance to cells. However, recent work has revealed that hydrogen peroxide can be utilized by organisms as a cellular signaling molecule. Hydrogen peroxide has the ability to react with proteins involved in signal transduction causing the activity of these proteins to be turned on or off. One such example is protein tyrosine phosphatase 1B (PTP1B) whose enzymatic activity is turned off by reacting with hydrogen peroxide. A cysteine residue in the active site of PTP1B is oxidized by hydrogen peroxide to form a sulfenic acid which then reacts with a neighboring amide nitrogen in the protein backbone to form a cyclic sulfenamide. The formation of this heterocycle causes PTP1B to lose its activity. In order to discover new proteins that are oxidatively regulated by hydrogen peroxide, chemical tools for selective detection of cyclic sulfenamide residues in cellular probing need to be developed. We describe results obtained using simple chemical models to identify reagents that have the ability to selectively tag protein derived sulfenamide residues.Life Sciences Undergraduate Research Opportunity Progra

The formation of cysteine-tyrosine crosslinks via a sulfenic acid intermediate [abstract]

Abstract only availableCysteine residues in proteins are readily oxidized to sulfenic acids. Sulfenic acids, in turn, can act as potent electrophiles that have been observed to form intrastrand protein crosslinks with neighboring amide or cysteine residues. Cysteine-tyrosine crosslinks have also been observed in proteins, but the mechanism(s) of their formation is not clear. In the work presented here we investigated the intramolecular reaction between a sulfenic acid and a tyrosine mimic. The results provide chemical evidence that sulfenic acids have the potential to forge intrastrand protein crosslinks with tyrosine residues in proteins.Life Sciences Undergraduate Research Opportunity Progra

Kinetics and mechanism of protein tyrosine phosphatase 1B inactivation by acrolein

Human cells are exposed to the electrophilic [alpha],[beta]-unsaturated aldehyde acrolein from a variety of sources. The reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well-characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B (kinact = 0.02 [plus or minus] 0.005 s-1 and KI = 2.3 [plus or minus] 0.6 x 10-4 M). The enzyme activity does not return upon gel filtration of the inactivated enzyme, and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together, these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon-carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally related α,β-unsaturated aldehydes

A new approach toward PTP-1B inhibition

Abstract only availableSignaling pathways for cellular metabolism, growth, proliferation, differentiation, immune response, motility, and tissue homeostasis is regulated by the phosphorylation of protein tyrosine residues on target proteins in the relevant signal transduction pathways. Phosphorylation levels of tyrosine residues are controlled by the opposing actions of two enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Protein tyrosine kinases add phosphoryl groups while PTPs catalyze their removal. PTPs are emerging as potential drug targets for the treatment of type 2 diabetes, autoimmune diseases, osteoporosis, and cancer. PTP-1B is the archetypal PTP and its inactivation may be a viable treatment for type 2 diabetes and obesity. PTP-1B is regulated by endogenous hydrogen peroxide (H2O2), which is a known cellular signaling agent. H2O2 oxidatively-inactivates PTP-1B, and its activity is regenerated by free thiols within the cell such as glutathione. In order to facilitate enzyme regeneration the aforementioned thiol must have access to the enzyme active site. We are testing the hypothesis that small molecules can inhibit thiol-mediated reactivation of redox-inactivated PTPs. Molecules with such a property would decrease the activity of target PTPs in cells, thus enhancing cellular response to external stimuli that act through receptor protein kinases.Life Sciences Undergraduate Research Opportunity Progra