α-VINYLLYSINE AND α-VINYLARGININE ARE TIME-DEPENDENT INHIBITORS OF THEIR COGNATE DECARBOXYLASES

(±)-α-Vinyllysine and (±)-α-vinylarginine display time-dependent inhibition of L-lysine decarboxylase from B. cadaveris, and L-arginine decarboxylase from E. coli, respectively. A complete Kitz-Wilson analysis has been performed using a modification of the Palcic continuous UV assay for decarboxylase activity

Synthesis of Higher α-Chlorovinyl and α-Bromovinyl Amino Acids: The Amino Protecting Group Determines the Reaction Course

N-Trifluoroacetyl α-vinyl amino esters are smoothly converted to the corresponding α-chlorovinyl or α-bromovinyl amino esters through the agency of phenyselenyl chloride or phenylselenyl bromide, respectively, followed by oxidation and pyrolysis. Exclusively the (E)-extemal halovinyl isomer and the internal halovinyl isomer are observed. The amino protecting group is a critical determinant of the reaction course (alkene addition vs. 5-exo-trig-like cyclization)

Mitochondrial Trafficking by Prohibitin-Kinesin-Myosin- Cadherin Complex in the Eye

Disruption of the mitochondrial-nuclear network leads to accelerated aging and age-related diseases, including age-related macular degeneration. The current study tested the hypothesis that mitochondrial morphology could be demonstrated quantitatively using a mathematic model and mitochondrial trafficking complex under stress conditions. To test our hypothesis, normal and aberrant mitochondria were examined quantitatively based on mitochondrial size, shape, position, composition, and dynamics. Adaptation of the mitochondrial network to changes in the intracellular oxidation and reduction milieu is critical for the survival of retinal pigment epithelial cells. Our mitochondrial interactome mapping demonstrated that a positive correlation may exist between oxidative stress-mediated phosphorylation and age-related disease progression. The current interactome may provide a potential therapeutic approach to treat mitochondria-induced neurodegeneration, including age-related macular degeneration

Mechanistic Dissection of Macular Degeneration Using the Phosphorylation Interactome

In the current study, we suggest that phosphorylation reactions of specific proteins in mitochondria and the nucleus are a key step in the progression of age-related macular degeneration (AMD). To determine the molecular mechanism of AMD, we examined proteomic changes under oxidative stress to establish the protein interaction map using in vitro and in vivo models that mimic the complex and progressive characteristics of AMD. We postulated that apoptosis can be initiated by phosphorylation reactions under chronic oxidative stress in a region-specific and tissue-specific manner. The analysis of AMD interactome and oxidative biomarker network demonstrated that the presence of tissue- and region-dependent post-translational mechanisms may contribute toward AMD progression through the mitochondrial-nuclear communication. The AMD interactome suggests that new therapeutic targets, including prohibitin, erythropoietin, vitronectin, crystalline, nitric oxide synthase, ubiquitin, and complement inhibition may exist as a proteome network. Further, immunocytochemistry demonstrated that mitochondria could enter the nucleus in the retinal pigment epithelium (RPE) under oxidative stress. The current interactome map implies that a positive correlation may exist between oxidative stress-mediated phosphorylation and AMD progression. The unbiased proteome network provides a basis for understanding oxidative stress-induced mitochondrial dysfunction in AMD and exploring effective therapeutic approaches to treat age-related neurodegeneration

Role of Rho-kinase in regulation of insulin action and glucose homeostasis

SummaryAccumulating evidence indicates an important role for serine phosphorylation of IRS-1 in the regulation of insulin action. Recent studies suggest that Rho-kinase (ROK) is a mediator of insulin signaling, via interaction with IRS-1. Here we show that insulin stimulation of glucose transport is impaired when ROK is chemically or biologically inhibited in cultured adipocytes and myotubes and in isolated soleus muscle ex vivo. Inactivation of ROK also reduces insulin-stimulated IRS-1 tyrosine phosphorylation and PI3K activity. Moreover, inhibition of ROK activity in mice causes insulin resistance by reducing insulin-stimulated glucose uptake in skeletal muscle in vivo. Mass spectrometry analysis identifies IRS-1 Ser632/635 as substrates of ROK in vitro, and mutation of these sites inhibits insulin signaling. These results strongly suggest that ROK regulates insulin-stimulated glucose transport in vitro and in vivo. Thus, ROK is an important regulator of insulin signaling and glucose metabolism

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle

Synthesis and evaluation of new alpha-halovinyl amino acids as amino acid decarboxylase inactivators

From the medicinal point of view, several amino acid decarboxylases (Arg DC, DOPA DC, Glu DC, His DC, Lys DC, Orn DC, SAM DC) are important targets for the development of specific and potent inactivators. Higher amino acid analogues, in which the α-hydrogen of amino acid is replaced by a chlorovinyl or fluorovinyl group, are potential mechanism-based enzyme inhibitors for their cognate amino acid decarboxylases. This dissertation describes, to our knowledge, the first syntheses and enzymatic evaluation of these two families of quaternary amino acids. Our synthetic approach to quaternary α-chloro and α-bromovinyl amino acids is based upon addition of the appropriate phenylselenenyl halide to suitably protected α-vinyl amino acids, followed by selenide oxidation and pyrolysis. Under conditions of kinetic control (PhSeX, MeCN, 2 ∼ 3 hours), primarily the (±)-(E)-α-(2-chlorovinyl) amino acids are obtained. At longer times for the PhSeX addition step, the (±)-α-(1-chlorovinyl) regioisomer is seen, comprising 50 ∼ 100% of the α-halovinyl amino acids mixture. The direct α-(1-chloro) vinylation using 2,2-dichlorovinyl phenyl sulfone was accomplished as a potential synthetic route to (±)-α-(1-chlorovinyl) amino acids. As proof of principle, free (±)-α-(1-chlorovinyl)alanine and (±)-α-(1-chlorovinyl)glutamate have been synthesized using this chemistry. A series of (±)-(Z)-α-(2-fluorovinyl) amino acids was synthesized from α-formyl amino acids via a Horner-Wadsworth-Emmons reaction with very high stereoselectivity. The resultant α-fluorovinyl sulfone functionality was transformed into an α-fluorovinyl stannane group with high stereoselectivity for all quaternary amino acids examined. Protodestannylation and deprotection were accomplished to give six (±)-(Z)-α-(2-fluorovinyl) amino acids (Ala, DOPA, Glu, Lys, Phe, m-Tyr). A coupled continuous UV assay was used to evaluate inhibitory effects of (±)-α-vinyllysine and (±)-α-vinylarginine. Time-dependent inhibition was observed for both compounds. (±)-(E)-α-(2-Chlorovinyl)lysine, (±)-α-vinyllysine, (±)-(Z)-α-(2-fluorovinyl)lysine, (±)-α-vinylglutamate, and (±)-(Z)-α-(2-fluorovinyl)glutamate were tested with their cognate amino acids decarboxylases with a radioactive time-point assay. Lineweaver-Burk analysis was used to test each assay and to obtain values of Km for each natural substrate (L-lysine/LDC Km = 1.9 mM, L-glutamate/GAD Km = 1.3 mM) and Kitz-Wilson analysis were used to obtain KI, kinact, and t 1/2. Extended dialysis showed the inhibition of lysine decarboxylase to be essentially irreversible. In the case of the best lysine decarboxylase inactivator found, (±)-(Z)-α-(2-fluorovinyl)lysine, non-pseudofirst order kinetics (ln (Et/Eo) vs. preincubation time) were observed. A Kitz-Wilson analysis of these data using tangents to the inactivation curves provided KI = 1.3 mM, kinact = 0.15 min–1 , t1/2 = 5 minutes. Consistent with the notion that this compound is a “suicide substrat”, the product of its enzymatic decarboxylation was observed by 19F NMR and the ratio of turnover product observed to total enzyme inactivated is approximately 46. That the observed 19F NMR signal was due to product has been confirmed by independent synthesis of (Z)-α-(2-fluorovinyl)cadaverine

Synthesis of Higher α-Chlorovinyl and α-Bromovinyl Amino Acids: The Amino Protecting Group Determines the Reaction Course

N-Trifluoroacetyl α-vinyl amino esters are smoothly converted to the corresponding α-chlorovinyl or α-bromovinyl amino esters through the agency of phenyselenyl chloride or phenylselenyl bromide, respectively, followed by oxidation and pyrolysis. Exclusively the (E)-extemal halovinyl isomer and the internal halovinyl isomer are observed. The amino protecting group is a critical determinant of the reaction course (alkene addition vs. 5-exo-trig-like cyclization)