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

Chemical Properties of the Leinamycin-Guanine Adduct in DNA

The reaction of the antitumor agent leinamycin with thiols converts this natural product into an episulfonium ion that alkylates the N7-position of guanine residues in double-stranded DNA. It is reported here that depurination of this adduct is unusually facile, occurring with a half-life of about 3.5 h at pH 7 and 37°C in duplex DNA. This is one of the most rapid depurination reactions ever observed for an N7-alkylguanine residue. The rate constant for the depurination reaction was measured at several temperatures, and the activation parameters were calculated from the data. The energy of activation (E a ) for this reaction is 24.6 kcal/mol, and the Arrhenius A value is 1.2 × 10 13 s -1 . These values correspond to a ∆H ‡ ) 24.0 kcal/mol and ∆S ‡ ) -0.78 eu and are consistent with the expected unimolecular (D N + A N ) mechanism for the depurination reaction. Changes in ionic strength (0-500 mM NaCl) or pH (3-8) do not significantly alter the rate of depurination, and the base excision repair protein Aag, which removes a variety of N7-alkylguanine residues from duplex DNA, does not excise the leinamycin-guanine adduct. Possible biological implications of this rapid depurination process are considered. Finally, during the course of these studies, the release of hydrolyzed leinamycin (4; Scheme 1) from leinamycin-modified DNA was observed. This result suggests that leinamycin may be a reversible DNA alkylating agent

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

Microlensing and Halo Cold Dark Matter

We discuss the implications of the more than 50 microlensing events seen by the EROS, MACHO, and OGLE collaborations for the composition of the halo of our galaxy. The event rates indicate that the halo mass fraction in MACHO's is less than 30\%, consistent with expectations for a universe whose primary component is cold dark matter. We caution that the uncertainties are such that a larger MACHO fraction cannot yet be excluded.Comment: 8 pages, latex, 4 figures, (Minor revisions of our galactic models

Spectroscopic Observations and Analysis of the Unusual Type Ia SN 1999ac

We present optical spectra of the peculiar Type Ia supernova (SN Ia) 1999ac. The data extend from -15 to +42 days with respect to B-band maximum and reveal an event that is unusual in several respects. Prior to B-band maximum, the spectra resemble those of SN 1999aa, a slowly declining event, but possess stronger SiII and CaII signatures (more characteristic of a spectroscopically normal SN). Spectra after B-band maximum appear more normal. The expansion velocities inferred from the Iron lines appear to be lower than average; whereas, the expansion velocity inferred from Calcium H and K are higher than average. The expansion velocities inferred from SiII are among the slowest ever observed, though SN 1999ac is not particularly dim. The analysis of the parameters v_10, R(SiII), dv(SiII)/dt, and d_m15 further underlines the unique characteristics of SN 1999ac. We find convincing evidence of CII 6580 in the day -15 spectrum with ejection velocity v > 16,000 km/s, but this signature disappears by day -9. This rapid evolution at early times highlights the importance of extremely early-time spectroscopy.Comment: 40 pages, 24 figures, accepted for publication in The Astronomical Journa

Spectroscopic observations and analysis of the peculiar SN 1999aa

We present an extensive new time series of spectroscopic data of the peculiar SN 1999aa in NGC 2595. Our data set includes 25 optical spectra between -11 and +58 days with respect to B-band maximum light, providing an unusually complete time history. The early spectra resemble those of an SN 1991T-like object but with a relatively strong Ca H and K absorption feature. The first clear sign of Si II λ6355, characteristic of Type Ia supernovae, is found at day -7, and its velocity remains constant up to at least the first month after B-band maximum light. The transition to normal-looking spectra is found to occur earlier than in SN 1991T, suggesting SN 1999aa as a possible link between SN 1991T-like and Branch-normal supernovae. Comparing the observations with synthetic spectra, doubly ionized Fe, Si, and Ni are identified at early epochs. These are characteristic of SN 1991T-like objects. Furthermore, in the day -11 spectrum, evidence is found for an absorption feature that could be identified as high velocity C II λ6580 or Hα. At the same epoch C II λ4648.8 at photospheric velocity is probably responsible for the absorption feature at 4500 Å. High-velocity Ca is found around maximum light together with Si II and Fe II confined in a narrow velocity window. Implied constraints on supernovae progenitor systems and explosion hydrodynamic models are briefly discussed