Inactivation of protein tyrosine phosphatases by endogenous and dietary agents

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 16, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Kent S. Gates.Vita.Ph. D. University of Missouri--Columbia 2009.Protein tyrosine phosphatases are a class of enzymes that control a number of critical signaling pathways inside cells. We have discovered a number of dietary and endogenous agents that are capable of modifying these enzymes, and therefore disrupting signaling pathways inside cells. It is possible that these small molecules are exerting their widely reported biological effect by the modification of protein tyrosine phosphatase activity. This information is critical to our understanding of dietary effect on diseases like diabetes and cancer. In addition to this work, we have explored the chemistry of the most common DNA lesion, the abasic site. We have found it is capable of producing an interstrand crosslink in duplex DNA, when placed in the right sequence context. This work significantly expands our understanding of the toxicity of abasic sites to cells. Furthermore, this work may result in developing novel treatment strategies for cancer.Includes bibliographical reference

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

Inactivation of PTP1B by endogenous and dietary aldehydes [abstract]

Abstract only availableFaculty Mentor: Kent S. Gates, ChemistryProtein tyrosine phosphatase-1B (PTP1B) plays a significant role in the tyrosine phosphorylation-dependent signal transduction by dephosphorylating the tyrosine residue in the insulin signaling pathway. PTP1B has a catalytic cysteine residue (Cys 215) at the active site. Knockout studies have shown that there is an enhancement of insulin sensitivity and obesity resistance in mice without the gene for PTP1B, making the enzyme a target for treatment of Type II diabetes. Efforts are ongoing in the search for the drug inhibitors of PTP-1B. It is also crucial to identify endogenous and/or dietary inhibitors of the enzyme. For instance, aldehydes are consumed in diet and generated by lipid metabolism. They are also known as inhibitors of cysteine dependent enzymes. Accordingly, we have studied acrolein, an unsaturated aldehyde, and investigated whether it can act as an endogenous /dietary inhibitor of PTP1B. Indeed, we find that acrolein inactivates PTP1B and is higher in potency than hydrogen peroxide, the known endogenous regulator of PTP1B activity. The inactivation of PTP1B by acrolein is irreversible. The inhibition is active site directed which is confirmed by the use of a competitive inhibitor phosphate. We find that simple aldehydes lacking conjugated double bonds are not inactivators of PTP1B. In conclusion, we can say that acrolein is a powerful inactivator of PTP1B and the inactivation of PTP1B proceeds through addition at the double bond not at the carbonyl group of acrolein.Protein tyrosine phosphatase-1B (PTP1B) plays a significant role in the tyrosine phosphorylation-dependent signal transduction by dephosphorylating the tyrosine residue in the insulin signaling pathway. PTP1B has a catalytic cysteine residue (Cys 215) at the active site. Knockout studies have shown that there is an enhancement of insulin sensitivity and obesity resistance in mice without the gene for PTP1B, making the enzyme a target for treatment of Type II diabetes. Efforts are ongoing in the search for the drug inhibitors of PTP-1B. It is also crucial to identify endogenous and/or dietary inhibitors of the enzyme. For instance, aldehydes are consumed in diet and generated by lipid metabolism. They are also known as inhibitors of cysteine dependent enzymes. Accordingly, we have studied acrolein, an unsaturated aldehyde, and investigated whether it can act as an endogenous /dietary inhibitor of PTP1B. Indeed, we find that acrolein inactivates PTP1B and is higher in potency than hydrogen peroxide, the known endogenous regulator of PTP1B activity. The inactivation of PTP1B by acrolein is irreversible. The inhibition is active site directed which is confirmed by the use of a competitive inhibitor phosphate. We find that simple aldehydes lacking conjugated double bonds are not inactivators of PTP1B. In conclusion, we can say that acrolein is a powerful inactivator of PTP1B and the inactivation of PTP1B proceeds through addition at the double bond not at the carbonyl group of acrolein