The role of initiator tRNAimet in fidelity of initiation of protein synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA(i) (met) compared to the one for the elongator tRNAs indicates that only the initiator tRNA(i) (met) specifies the amino acid side chain.     

Expression analysis and molecular targeting of cyclin-dependent kinases in advanced melanoma: Functional analysis and molecular targeting of cyclin-dependent kinase family members in advanced melanoma

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1 and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.Key words: melanoma, cyclin-dependent kinase expression, small-molecule inhibitor treatment, proliferation, cell cycle progression, apoptosis, tumor xenograft studies     

Comparing Trends in BMI and Waist Circumference

The nature of excess body weight may be changing over time to one of greater central adiposity. The aim of this study is to determine whether BMI and waist circumference (WC) are increasing proportionately among population subgroups and the range of bodyweight, and to examine the public health implications of the findings. Our data are from two cross‐sectional surveys (the US National Health and Nutrition Examination Studies (NHANES) in 1988–1994 (NHANES III) and 2005–2006), from which we have used samples of 15,349 and 4,176 participants aged ≥20 years. Between 1988–1994 and 2005–2006 BMI increased by an average of 1.8 kg/m² and WC by 4.7 cm (adjusted for sex, age, race‐ethnicity, and education). The increase in WC was more than could be attributed simply to increases in BMI. This independent increase in WC (of on average, 0.9 cm) was consistent across the different BMI categories, sexes, education levels, and race‐ethnicity groups. It occurred in younger but not older age groups. Overall in each BMI category, the prevalence of low‐risk WC decreased and the prevalence of increased‐risk or substantially increased‐risk WC increased. These results suggest that the adverse health consequences associated with obesity may be increasingly underestimated by trends in BMI alone. Since WC is closely linked to adverse cardiovascular outcomes, it is important to know the prevailing trends in both of these parameters.     

Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases

The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1–4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.Neoplastic transformation and growth require a massive increase in glucose uptake and glycolytic flux not only for energy production but also for the synthesis of nucleic acids, amino acids, and fatty acids. A central control point of glycolysis is the negative allosteric regulation of a rate-limiting enzyme, phosphofructokinase-1 (PFK-1),² by ATP (i.e. the Pasteur effect) (1, 2). When intracellular ATP production exceeds usage, ATP inhibits PFK-1 and glycolytic flux. Fructose 2,6-bisphosphate (Fru-2,6-BP) is a potent allosteric activator of PFK-1 that overrides this inhibitory influence of ATP on PFK-1, allowing forward flux of the entire pathway (3–5).The steady-state cellular concentration of Fru-2,6-BP is dependent on the activities of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four independent genes (PFKFB1–4) (6, 7). The PFKFB3 mRNA is distinguished by the presence of multiple copies of an AUUUA instability motif in its 3′-untranslated region and the PFKFB3 protein product has a high kinase:phosphatase activity ratio (740:1) (8). PFKFB3 mRNA is overexpressed by rapidly proliferating transformed cells and the PFKFB3 protein is highly expressed in solid tumors and leukemias (8–11). PFKFB3 expression is increased in response to several mitogenic stimuli, including progesterone, serum, and insulin (12–14). These studies indicate that the PFKFB3 enzyme may serve an essential function in the regulation of glucose metabolism during cell proliferation.The PFKFB3 mRNA is spliced into several variants that encode distinct carboxyl-terminal domains (9, 15). Importantly, the functional consequences of the disparate carboxyl-terminal variants of PFKFB3 are unknown. The mRNA splice variant 5 is the dominant PFKFB3 mRNA in human brain, several transformed cells, and colon adenocarcinoma tissues (9, 10). In the following series of experiments, we present data that the carboxyl-terminal domain of PFKFB3 variant 5 localizes the enzyme to the nucleus where its product, Fru-2,6-BP, increases the expression and activity of cyclin-dependent kinase-1. These data demonstrate a heretofore unidentified function of the PFKFB3 enzyme that is distinct from glycolysis, and provide a potential mechanism for the coupling of metabolism and proliferation.