Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.

Project description:Background: A significant body of evidence accumulated over the last century suggests a link between hypoxic microenvironments within the infected host and the latent phase of tuberculosis. Studies to test this correlation have identified the M. tuberculosis initial hypoxic response, controlled by the two-component response regulator DosR. The initial hypoxic response is completely blocked in a dosR deletion mutant. Methodology/Principal Findings: We show here that a dosR deletion mutant enters bacteriostasis in response to in vitro hypoxia with only a relatively mild decrease in viability. In the murine infection model, the phenotype of the mutant was indistinguishable from that of the parent strain. These results suggested that additional genes may be essential for entry into and maintenance of bacteriostasis. Detailed microarray analysis of oxygen starved cultures revealed that DosR regulon induction is transient, with induction of nearly half the genes returning to baseline within 24 hours. In addition, a larger, sustained wave of gene expression follows the DosR-mediated initial hypoxic response. This Enduring Hypoxic Response (EHR) consists of 230 genes significantly induced at four and seven days of hypoxia but not at initial time points. These genes include a surprising number of transcriptional regulators that could control the program of bacteriostasis. We found that the EHR is independent of the DosR-mediated initial hypoxic response, as EHR expression is virtually unaltered in the dosR mutant. Conclusions/Significance: Our results suggest a reassessment of the role of DosR and the initial hypoxic response in MTB physiology. Instead of a primary role in survival of hypoxia induced bacteriostasis, DosR may regulate a response that is largely optional in vitro and in mouse infections. Analysis of the EHR should help elucidate the key regulatory factors and enzymatic machinery exploited by M. tuberculosis for long-term bacteriostasis in the face of oxygen deprivation. Keywords: time-course Each hypoxic timecourse compared to the matching log phase sample. Each time point/strain represented by a minimum of three replicates.

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Glycolytic Enzyme Coalesce in G bodies Under Hypoxic Stress

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