<p> The development of metabolic dysfunctions like diabetes and insulin resistance in mammals is regulated by a myriad of factors. Oxidative stress seems to play a central role in this process as recent evidence shows a general increase in oxidative damage and a decrease in oxidative defense associated with several metabolic diseases. These changes in oxidative stress can be directly correlated with increased fat accumulation, obesity and consumption of high calorie/high fat diets. Modulation of oxidant protection through either genetic mutation or treatment with antioxidants can significantly alter oxidative stress resistance and accumulation of oxidative damage in laboratory rodents. Antioxidant mutant mice have previously been utilized to examine the role of oxidative stress in other disease models, but have been relatively unexplored as models to study the regulation of glucose metabolism. </p><p> Our studies have focused on MsrA in mammalian systems and have utilized mice that lack MsrA (<i>MsrA^-/-</i>) and that over express MsrA (MsrA^mitoTg and MsrA^cytoTg). Under normal conditions, our lab has shown that <i>MsrA^-/-</i> mice are phenotypically similar to wildtype (WT) mice, but are susceptible to oxidative stress [244]. MsrA^mitoTg and MsrA^cytoTg are also phenotypically similar to WT (though oxidative stress resistance has not yet been tested). This indicates that excess methionine oxidation may not occur at basal ROS levels, which is supported by yeast studies [202]. In vivo, increasing adiposity has been associated with increases in oxidative stress, altered redox signaling and increased oxidative damage to cellular macromolecules in several disease models. It is also thought that adipose tissue-induced oxidative stress may be a primary factor in the etiology of obesity-induced metabolic diseases. When placed on a high fat (HF) diet to induce an increase in oxidative stress, <i>MsrA^-/-</i> mice become more insulin resistant than WT mice whereas MsrA^mitoTg mice are protected. The increase in insulin resistance in <i> MsrA^-/-</i> mice is not due to further exacerbation of pathways thought to link oxidative stress and insulin resistance (JNK signaling or pro-inflammatory cytokine expression). These results suggest that oxidative damage to proteins may play an important role in obesity-induced insulin resistance. </p>

<p> Sphingolipids (SL) are essential components of eukaryotic cells involved maintaining membrane integrity. They are important for membrane trafficking and function in signaling as messenger molecules. SLs are synthesized <i> de novo</i> from very long chain fatty acids (VLCFA) and sphingoid long-chain bases (LCB), which are amide-linked to form ceramide and further processed by addition of various head-groups. Little is known concerning the regulation of VLCFA levels and how cells coordinate their synthesis with the availability of LCBs for SL synthesis. Here I show that Elo2, a key enzyme of VLCFA synthesis, is controlled by signaling of the guanine nucleotide exchange factor Rom2, initiating at the plasma membrane. This pathway controls Elo2 phosphorylation state and VLCFA synthesis. My data identify a regulatory mechanism for coordinating VLCFA synthesis with SL metabolism and link signal transduction pathways from the plasma membrane to the regulation of lipids for membrane homeostasj.</p>

<p> In order to adapt to changing conditions and life stages, cells must remodel the composition of the proteome by specifically removing obsolete or damaged proteins. Failure to remove proteins at the appropriate time can cause a variety of cellular dysfunctions that can lead to cell death and conditions such as cancer, Huntington's, Parkinson's and Alzheimer's disease. Protein quality control is especially important during aging and stress, as the accumulating load of damaged, misfolded, and aggregated proteins can overwhelm the cell's ability to maintain the proteome. The ubiquitin-proteasome system is responsible for the specific degradation of many proteins within eukaryotic cells. The 26S proteasome degrades proteins that have been &ldquo;tagged&rdquo; with a chain of ubiquitin molecules. Ubiquitin is a highly-conserved 8.5 kDa protein that is conjugated post-translationally to a target protein after passing through a complex series of enzymes. Using the nematode <i> C. elegans</i> as a model, we sought to 1) Characterize the roles of specific ubiquitin-conjugation enzymes (UBCs) in the response to aggregation of a polyglutamine-containing protein in body wall muscle cells and 2) Examine the subcellular localization of proteasomes in these cells during normal aging and stress. Q82::GFP, a fusion protein expressed from a transgene encoding a polyglutamine tract fused to Green Fluorescent Protein (GFP), was found to aggregate rapidly (~58 minutes) in a manner that is not directly dependent on ubiquitin but dependent on the concentration of Q82::GFP protein, which was altered in response to RNA interference of several E2 ubiquitin-conjugating enzymes. In a separate transgenic worm strain, a fluorescently-labeled proteasome subunit, RPT-1, was observed in live animals to localize to the nucleus and cytoplasm during the adult stage. Within the nucleus, RPT-1::GFP localizes diffusely and to foci, and was excluded from heterochromatin. Within the muscle contractile apparatus, RPT-1::GFP localizes to I-bands, regions of thin actin filaments, and is excluded from dense bodies. After a prolonged heat stress, RPT-1::GFP in adult worms relocalizes to dense bodies. After starvation, these foci are associated with high proteolytic activity. These studies demonstrate some of the spatial dynamics of the ubiquitin-proteasome system in response to development, aging, and stress.</p>

<p> Progression through the mammalian cell cycle is a tightly regulated process that allows cells to replicate their genomes and divide properly. In growth factor-deprived conditions or in response to stress, the cell will exit the cell cycle either reversibly through quiescence, or permanently via senescence. Studies have shown that the SIN3 family of proteins plays a crucial role in these cell cycle exit processes. SIN3 proteins are highly conserved, and exist in mammals as two family members: SIN3A and SIN3B, which function as flexible scaffolding proteins to assemble co-repressor complexes. Our laboratory has recently implicated SIN3B as a critical mediator of each of these cell cycle exit processes. However, its mechanism of action and the consequences of its disruption pertaining to cancer progression have not been comprehensively elucidated. Here we demonstrate that SIN3B is required for the induction of senescence in a mouse model of prostate cancer, and thus prevents the progression to aggressive and invasive carcinoma. In addition, through interaction analysis, we uncovered a novel and robust association between SIN3B and the DREAM complex. The DREAM complex, comprised of p107/p130, E2F4/5, DP1 and the MuvB core complex, is responsible for the repression of hundreds of cell cycle-related transcripts during quiescence. We determined that the deletion of <i>SIN3B</i> resulted in the derepression of DREAM target genes during quiescence, but was not sufficient to allow quiescent cells to resume proliferation. However, the ectopic expression of APC/C^CDH1 inhibitor EMI1 was sufficient for <i>SIN3B</i> deleted cells, but not wild-type cells, to reenter the cell cycle. These studies demonstrate a critical role for SIN3B in the senescence and quiescence programs, and provide important mechanistic insight into the molecular pathways that exquisitely regulate cell cycle exit.</p><p>

<p> This study examined various hybrid peptides that possess both collagen [(POG)_n] and cell penetrating peptides (CPP) [(RRG)_n or R_n] sequences. The hybrid peptides were able to fold into triple helical conformation when the surrounding temperature was lower than their transition temperature (T_m) which resulted in cellular internalization. The peptide that lacked collagen [(POG)_n] domain failed to penetrate the cell. The hybrid peptide under study, FL7V1, was shown to have the ideal T_m (17.3&deg;C) for the potential purpose as a drug carrier. <i>In vitro</i> study of FL6V1 with temperature gradient showed cellular internalization at low temperatures (10&deg;C-20&deg;C) while no uptake was achieved at high temperatures (24&deg;C-32&deg;C). <i> In vivo</i> study of FL7V1 with <i>P. leidyi</i> corresponded with the results of <i>in vitro</i> study at constant and gradient temperature.</p>

The antioxidant activities of compounds endogenous to bovine rod outer segments (ROS) were investigated by measuring the loss of polyunsaturated fatty acids (PUFA's) from membranes exposed to the water-soluble oxidant $2,2\sp\prime$-azobis(2-amidinopropane) dihydrochloride (AAPH). Osmotically intact ROS, ROS membranes, and unilamellar liposomes prepared from ROS phospholipids (PL) were compared. Intact ROS were most resistant to oxidative loss of PUFA's, followed by ROS membranes and then PL liposomes. The development of a model membrane system allowed the investigation of putative antioxidants singly and in combination. Lipid-soluble compounds (vitamin E, free fatty acids, retinol, retinaldehyde) were incorporated into PL liposomes. Water-soluble compounds (reduced glutathione, taurine) were dissolved in incubation buffer. It was found that normal physiological concentrations of alpha-tocopherol (vitamin E), free fatty acids (16:0, 18:0, 18:1, 22:6), retinol, and glutathione significantly decreased oxidative loss of PUFA's. When the major free fatty acids were added to PL liposomes at the same concentrations found when ROS phospholipase A is stimulated, the oxidative loss of PUFA's was reduced by 31%. The antioxidant effect of free fatty acids suggests that endogenous phospholipase A's may act to protect membranes by releasing fatty acids from phospholipids in proportions and concentrations that afford protection to membrane lipids. The antioxidant activities of the two major retinoid compounds involved in the visual cycle in ROS, all-trans retinaldehyde and all-trans retinol, were compared. The addition of retinol partially protected PUFA's in ROS PL liposomes, whereas retinaldehyde promoted lipid peroxidation. When isolated ROS were stimulated to produce endogenous retinol, PUFA loss was inhibited by up to 17%. These findings suggest that there is an antioxidant function for the enzymatic reduction of retinaldehyde to retinol during photoreception. Water-soluble antioxidants, taurine and reduced glutathione (GSH), were investigated individually and in combination with retinol in ROS PL liposomes. GSH significantly protected PUFA's in ROS PL liposomes. Taurine alone showed little antioxidant activity, but in combination with retinol protected lipids by up to 47.9% (an increase in antioxidant protection of 24.8% over retinol alone). These data support previous findings that taurine protects ROS lipids during exposure to cyclic light conditions.

N-linked glycans serve as quality control tags in the eukaryotic secretory pathway. The endoplasmic reticulum (ER) protein UDP-glucose: glycoprotein glucosyltransferase 1 (GT1) is the main enzyme that modifies carbohydrate tags based upon the folding state of the maturing substrate. GT1 adds glucoses to non-glucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate as well as its ability to differentiate between native or aberrant secretory cargo is poorly understood. Additionally, due to inherent difficulties in studying GT1 activity in the cell, identification of endogenous substrates and the necessity of reglucosylation remain unknown. Here, we analyzed the role of GT1 in glycoprotein maturation in the intact mammalian ER. GT1 post-translationally reglucosylates N-linked glycans in slow-folding regions of substrate glycoproteins. Maturation mutants that disrupt oxidation or oligomerization also support regio-specific reglucosylation by GT1. Our studies have also revealed an abundant endogenous substrate of GT1, identified as prosaposin. GT1 is critical for the maturation of endogenous prosaposin. In the absence of GT1, the endogenous protein is mislocalized to large intracellular juxtanuclear aggregates. Together, these results propose that GT1 acts as an ER quality control sensor by post-translationally targeting glycans on slow folding or non-native domains to recruit chaperones specifically to critical unstable regions. GT1 plays a vital role in endogenous protein folding and trafficking, since in its absence misfolded proteins accumulate intracellularly. This investigation provides new insight into the integral role of GT1 in glycoprotein maturation.

Human lysosomal α-N-acetylgalactosaminidase (α-NAGAL) is responsible for the break down of glycolipids and glycopeptides that contain a terminal α-linked N-acetylgalactosamine residues. Deficiency of α-NAGAL results in Schindler and Kanzaki diseases. α-NAGAL is closely related to another lysosomal enzyme, α-galactosidase (α-GAL), which breaks down glycolipids and glycopeptides with a terminal α-linked galactose residues. Fabry disease results from a deficiency of α-galactosidase activity. We studied the reaction mechanism of both enzymes using biochemistry and X-ray crystallography, and found that α-GAL and α-NAGAL use an identical reaction mechanism, and differ only in substrate specificity. We solved the first structure of human α-NAGAL, allowing us to examine the disease-causing patient mutations in the context of a high-resolution 3D atomic structure, moving Schindler and Kanzaki disease into the realm of personalized molecular medicine. We then developed the first ever proof-of-principle treatment of Schindler and Kanzaki disease, by developing and characterizing 2 pharmacological chaperones that show promise to treat Schindler and Kanzaki diseases, which currently have no treatment options.

Viruses have evolved to exploit cellular pathways and machinery in order to deliver their genome to the cell, replicate, and produce viral progeny. Nonenveloped viruses must overcome membrane barriers to infect host cells and trigger lysis for virion release. The model nonenveloped virus, Simian Virus 40 (SV40), is bound at the cell surface and eventually delivered to the endoplasmic reticulum (ER) where penetration occurs resulting in delivery of the viral genome to the nucleus by an unknown mechanism. During the later stages of infection viral progeny are assembled in the nucleus and are liberated from the host cell through a cytolytic process. SV40 appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection for virus release. Bacterially expressed and purified VP4 forms size selective pores in membranes. To investigate the role of VP4 in host cell lysis an inducible expression system was used to produce VP4 in mammalian cells. The viral protein was mainly localized along the nuclear envelope and correlated with the mislocalization of nuclear proteins and was associated with cell death. These results indicate that VP4 acts as a viroporin in the nuclear membrane to promote virus release. Previous results indicated that the two minor structural proteins, VP2 and VP3, may act as membrane proteins during viral infection. Studies using purified proteins, bioinformatics, a cell-free membrane insertion assay and a thorough examination of viral propagation, assembly and infection processes have provided new insights into the role of the minor structural proteins during infection. Targeted disruption of the viroporin activity of VP2 and VP3 inhibited viral infection. Together, these results support that the late viral proteins VP2, VP3 and VP4 each act as viroporins and serve as critical triggers for the progression of the viral life cycle. This investigation provides new insight into how the viroporin activity of the late viral proteins is utilized in viral infection and release.

The oxidation behavior of cholesterol and the relationship between cholesterol and non-cholesterol lipids in oxidation processes were studied in a bovine red blood cell membrane system (RBC Mb). RBC Mb was prepared with a process of hemolysis and hypotonic washing. After preparation, the RBC Mb suspensions were subjected to oxidation treatments of $\rm H\sb2O\sb2$-Fe, UV (254 nm, 312 nm, and 365 nm), and $\gamma$-irradiation. After the treatments, lipids were extracted, and cholesterol, cholesterol oxides and fatty acids were analyzed with GC and GC-MS. Of all the oxidation products, 7-keto-cholesterol was found to be the major oxidation product of cholesterol. Other oxidation products, to a less extent, were 7-hydroxylcholesterol, $\alpha$- and $\beta$-epoxycholesterol, etc. Oxidative destruction of cholesterol was detected together with the destruction of unsaturated and saturated fatty acids, although the destruction rates were different. Model studies indicated that cholesterol and fatty acids in the suspension of RBC Mb lipid extracts were much more susceptible to oxidative destruction than the cholesterol and fatty acids in the native RBC membranes. UV studies showed the complexity of cholesterol behavior in response to UV lights. Although the destruction of solid crystalline cholesterol at 312 nm UV went much faster than that at 254 nm and 365 nm, the fastest cholesterol destruction in RBC Mb aqueous suspensions occurred at 365 nm UV, which was the least powerful of the 3 wavelengths for solid crystalline cholesterol destruction. For 7-keto-cholesterol, the most destructive wavelength was 254 nm. In $\gamma$-irradiation study, there was no distinctive radiolytic product detected in this study. Contrasted with UV and $\rm H\sb2O\sb2$-Fe treatments, the destruction of cholesterol with $\gamma$-irradiation gave rise to far less production of cholesterol oxides which were detectable with the GC-MS procedures.