On vector-valued twisted conjugation invariant functions on a group; with an appendix by Stephen Donkin

We study the space of vector-valued (twisted) conjugate invariant functions on a connected reductive group

Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

Background: Proline is an important amino acid for stress resistance in different organisms. Results: Depletion of proline biosynthesis disrupts redox homeostasis and increases sensitivity to endoplasmic reticulum (ER) stress in yeast. Conclusion: Proline biosynthesis is critical for maintaining the intracellular redox environment and the UPR during ER stress. Significance: Proline metabolism is shown to have an important role in ER stress tolerance that was previously unknown

On vector-valued twisted conjugation invariant functions on a group; with an appendix by Stephen Donkin

We study the space of vector-valued (twisted) conjugate invariant functions on a connected reductive group

Targeted absolute quantitative proteomics with SILAC internal standards and unlabeled full‐length protein calibrators (TAQSI)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116918/1/rcm7482.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/116918/2/rcm7482_am.pd

Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins

Background: Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative. Results: We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity. Conclusion: Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation

Association of Oseltamivir Activation with Gender and Carboxylesterase 1 Genetic Polymorphisms

Oseltamivir, an inactive antiâ influenza virus prodrug, is activated (hydrolysed) in vivo by carboxylesterase 1 (CES1) to its active metabolite oseltamivir carboxylate. CES1 functions are significantly associated with certain CES1 genetic variants and some nonâ genetic factors. The purpose of this study was to investigate the effect of gender and several CES1 genetic polymorphisms on oseltamivir activation using a large set of individual human liver samples. CES1â mediated oseltamivir hydrolysis and CES1 genotypes, including the G143E (rs71647871), rs2244613, rs8192935, the â 816A>C (rs3785161) and the CES1P1/CES1P1VAR, were determined in 104 individual human livers. The results showed that hepatic CES1 protein expression in females was 17.3% higher than that in males (p = 0.039), while oseltamivir activation rate in the livers from female donors was 27.8% higher than that from males (p = 0.076). As for CES1 genetic polymorphisms, neither CES1 protein expression nor CES1 activity on oseltamivir activation was significantly associated with the rs2244613, rs8192935, â 816A>C or CES1P1/CES1P1VAR genotypes. However, oseltamivir hydrolysis in the livers with the genotype 143G/E was approximately 40% of that with the 143G/G genotype (0.7 ± 0.2 versus 1.8 ± 1.1 nmole/mg protein/min, p = 0.005). In summary, the results suggest that hepatic oseltamivir activation appears to be more efficient in females than that in males, and the activation can be impaired by functional CES1 variants, such as the G143E. However, clinical implication of CES1 gender differences and pharmacogenetics in oseltamivir pharmacotherapy warrants further investigations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134982/1/bcpt12625.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134982/2/bcpt12625_am.pd

Proline Mechanisms of Stress Survival

Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response

Proline dehydrogenase is essential for proline protection against hydrogen peroxide induced cell death

Proline metabolism has an underlying role in apoptotic signaling that impacts tumorigenesis. Proline is oxidized to glutamate in the mitochondria with the rate limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53 leading to increased proline oxidation, reactive oxygen species (ROS) formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and retained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress whereas knockdown of Δ1-pyrroline-5- carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the fork head transcription factor class O3a (FoxO3a). The role of PRODH in proline mediated protection was validated in the prostate carcinoma cell line, PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide mediated cell death and that proline/PRODH helps activate Akt in cancer cells