[Show abstract][Hide abstract] ABSTRACT: Recent studies highlight the importance of glutamine metabolism in metabolic reprogramming, which underlies cancer cell addiction to glutamine. Examples for the dependence on glutamine metabolism are seen across different tumor types as during different phases of cancer development, progression and response to therapy. In this perspective, we assess the possibility of targeting glutamine metabolism as a therapeutic modality for cancer.
[Show abstract][Hide abstract] ABSTRACT: Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth. A quantitative audit of how carbon from glutamine is used showed that TCA-cycle-derived glutamate is, in most melanoma cells, the major glutamine-derived cataplerotic output and product of glutaminolysis. In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion. Aspartate was an essential cataplerotic output, as melanoma cells demonstrated a limited capacity to salvage external aspartate. Also, the absence of asparagine increased the glutamine requirement, pointing to vulnerability in the aspartate-asparagine biosynthetic pathway within melanoma metabolism. In contrast to melanoma cells, melanocytes could grow in the absence of glutamine. Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.
[Show abstract][Hide abstract] ABSTRACT: 13C enrichment in proline and glutamate in Lu1205 cells at different time points. Cells were fed with [U-13C] glutamine in the presence of 0.3 mM of exogenous proline. Measurements of isotopic enrichment in proline and glutamate were made 2, 4, 6 and 8 hr after labeling. Results represent technical duplicates and standard deviations are less than 10%. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: 13C enrichment in proline in Lu1205 cells labeled with [U-13C] glutamine (8 h) in the presence of 0.5 mM of proline in the media. m0 is m/z 258 fragment ion, m1 is m/z 259, etc. Enrichment in proline is calculated with the following formula: Data represent average of two biological replicates and standard deviations are less than 5%. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: Kinetic characterization of recombinant PYCRs. Apparent kinetic parameters of human PYCRs with respect to substrate (A) and cofactors (B) were determined by monitoring the turnover of co-factors (NADH or NADPH). Results from steady-state kinetics experiments, obtained at saturating concentrations of cofactors*** and substrate*** (P5C) were graphed and fit with non-linear regression**. Apparent catalytic efficiencies of human PYCRs with respect to P5C and cofactors (C) were calculated using the apparent kcat and Km values presented in A and B. Apparent constants of PYCR inhibition by proline at saturating concentrations of co-factors (D) (1 mM NADH for PYCR1 and PYCR2 and 1.25 mM NADPH for PYCRL) were determined by fitting the steady-state kinetics data of P5C conversion to proline at varying concentrations of inhibitor with competitive inhibition equation. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13)C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: 13C enrichment in glutamate in Lu1205 cells labeled with [U-13C] glutamine (8 h) in the presence of 0.5 mM of proline in the media. m0 is m/z 330 fragment ion, m1 is m/z 331, etc. Enrichment in glutamate is calculated with the following formula: Data represent average of two biological replicates and standard deviations are less that 5%. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: Melanoma cells express higher levels of P5CS but similar levels of OAT compared to primary melanocytes. Expression of P5CS and OAT in melanocytes relative to melanoma cells represented by a panel of ten cell lines was determined by Western blotting. Specificities of the antibodies used are indicated on the left of each panel. β-actin was used as loading control. (TIFF)
[Show abstract][Hide abstract] ABSTRACT: Effect of PYCRs silencing in UACC903, WM1346 and WM1366 cells. The production of proline from glutamate, expressed as the enrichment ratio (pro/glu), was measured in cells labeled for 8 hr with [U-13C] glutamine (1 mM) in the presence of 0.3 mM of proline in the medium. Error bars represent standard deviations of biological duplicate. (TIFF)
[Show abstract][Hide abstract] ABSTRACT: Knockdown of PYCRs is without effect on isotopic enrichment in TCA cycle metabolites. Lu1205 cells were fed with [U-13C] glutamine in the presence of 0.5 mM of exogenous proline and isotopic enrichment was calculated after 8 hr of labeling. Data represent average of biological duplicates and standard deviations are less than 5%. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: Relative contribution of PYCRs to glutamate pathway. (A) Fraction of proline derived from glutamate expressed as the ratio of 13C enrichment of proline (product) over 13C enrichment of glutamate (precursor), measured upon silencing of PYCR1, PYCR2, PYCRL and P5CS. Lu1205 cells were labeled with [U-13C] glutamine (1 mM) for 8 h in the presence of 0.1, 0.3 and 0.5 mM of proline in the medium. (B) The same data are presented as % of change relative to non-specific siRNA (NS) control. Data are representative of two biological replicates and standard deviations are less than 5%. At 0.3 and 0.5 mM of exogenous proline, the data are consistent and show the same trend (as for PYCRL); at low concentration of proline (0.1 mM) there is not enough contribution of the salvage pathway to allow appreciable differences compared to the NS. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: Relative contribution of PYCRs to ornithine pathway. (A) Fraction of proline derived from ornithine expressed as the ratio of 13C enrichment of proline (product) over 13C enrichment of ornithine (precursor), measured upon silencing of PYCR1, PYCR2, PYCRL and P5CS. Lu1205 cells were labeled with [U-13C] ornithine (1 mM) for 8 h in the absence of proline in the medium. (B) The same data are presented as % of change relative to non-specific siRNA (NS) control. Data are representative of two biological replicates and standard deviations are less than 5%. (DOCX)
[Show abstract][Hide abstract] ABSTRACT: In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not 'dysfunction' but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non-conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. Biosynthetic networks linked with non-essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source.
[Show abstract][Hide abstract] ABSTRACT: Limited or regulatory proteolysis plays a critical role in many important biological pathways like blood coagulation, cell proliferation, and apoptosis. A better understanding of mechanisms that control this process is required for discovering new proteolytic events and for developing inhibitors with potential therapeutic value. Two features that determine the susceptibility of peptide bonds to proteolysis are the sequence in the vicinity of the scissile bond and the structural context in which the bond is displayed. In this study, we assessed statistical significance and predictive power of individual structural descriptors and combination thereof for the identification of cleavage sites. The analysis was performed on a data set of >200 proteolytic events documented in CutDB for a variety of mammalian regulatory proteases and their physiological substrates with known 3D structures. The results confirmed the significance and provided a ranking within three main categories of structural features: exposure > flexibility > local interactions. Among secondary structure elements, the largest frequency of proteolytic cleavage was confirmed for loops and lower but significant frequency for helices. Limited proteolysis has lower albeit appreciable frequency of occurrence in certain types of β-strands, which is in contrast with some previous reports. Descriptors deduced directly from the amino acid sequence displayed only marginal predictive capabilities. Homology-based structural models showed a predictive performance comparable to protein substrates with experimentally established structures. Overall, this study provided a foundation for accurate automated prediction of segments of protein structure susceptible to proteolytic processing and, potentially, other post-translational modifications.
Full-text · Article · Jun 2011 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions.
Full-text · Article · Feb 2009 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The Proteolysis MAP (PMAP, http://www.proteolysis.org) is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways.
PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB,
are driven by an automated annotation pipeline that generates dynamic ‘Molecule Pages’, rich in molecular information. PMAP
also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events,
and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within
these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically
modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease
Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be
used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis.
Full-text · Article · Nov 2008 · Nucleic Acids Research