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First published online July 23, 2003

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Evolution of the coordinate regulation of glycolytic enzyme genes by hypoxia

Keith A. Webster

Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, Miami, FL 33136, USA e-mail: kwebster{at}chroma.med.miami.edu

View larger version (18K): [in a new window]   Fig. 1. Composition and regulation of the glycolytic pathway in higher animals. (A) Linear pathway of glycolytic enzymes showing substrate input and sites of ATP utilization and generation. (B) Induction of glycolytic enzyme mRNA levels by hypoxia. Skeletal myocytes were exposed to hypoxia and mRNA transcript levels were measured at the indicated time points by the nuclear run-on technique, described in Webster (1987). The figure is a composite of six glycolytic enzyme gene transcripts using cDNAs to the enzymes indicated by the asterisks in A.

View larger version (67K): [in a new window]   Fig. 2. Southern blots illustrating strong conservation of glycolytic enzyme gene sequences across species. Cells or tissues from the indicated organisms were lysed and genomic DNA was extracted by standard techniques (Webster, 1987; Webster et al., 1990; Lonberg and Gilbert, 1985). DNA was digested with restriction enzyme EcoRI, separated on agarose gels and blotted onto nitrocellulose. Membranes were probed with 32P-cDNAs coding for pyruvate kinase (PK) and lactate dehydrogenase (LDH) as described in Webster (1987). Arrows indicate conserved DNA fragments. Note the increased number of hybridization bands for both genes in rodents (blocks indicated by vertical bars) that probably represent increased numbers of pseudogenes in these species (see text).

View larger version (18K): [in a new window]   Fig. 3. Milestones in evolution. (A) Paleontological periods of the Precambrian era. (B) Estimates of total earth biomass as a function of time. The graph is only a qualitative representation because it is not possible to establish or extrapolate precise levels of precambrian biomass from paleontological records (Kelly and Adams, 1994; Papagiannis, 1984; DeLong et al., 1994; Koch, 1998; O'Callaghan and Conrad, 1992; Reeve, 1992). BYA, billion years ago; Ph indicates initiation of the major increase in photosynthesis by cyanobacteria.

View larger version (114K): [in a new window]   Fig. 4. The Archean Age. (A) Stromatolite field as it may have looked 3.5 BYA (see text for details). (B) Stromatolite fossil; the arrows indicate `carbon films' where microscopic details reveal microbial fossils dating back to the earliest life forms on earth. (From the University of California at Berkeley Paleontological Museum; with permission).

View larger version (16K): [in a new window]   Fig. 5. Regulation of gene expression by redox-sensitive Arc and Fnr pathways. Under aerobic conditions, ArcB and ArcA are sequentially activated by phosphorylation. ArcA negatively regulates the transcription of the cyoABCDE operon, which encodes cytochrome bo oxidase (high Vmax, low oxygen affinity) and positively regulates cydAB encoding cytochrome bd oxidase (low Vmax, high oxygen affinity). Fnr is inactive under aerobic conditions, but at low oxygen it undergoes a conformational change, probably mediated by reduction of ferric to ferrous iron at an iron-sulphur center. Conformationally activated Fnr binds DNA at sites with the inverted repeat sequences TTGAT —— ATCAA. Fnr binding represses cyoABCDE and cydAB operons and induces transcription from the operons dmsABC (dimethyl sulfoxide/trimethylamine-N-oxide reductase), frdABCD (fumarate reductase), and narGHJI (nitrate reductase). Cross-talk between the two pathways at cyoABCDE and cydAB is indicated by the broken arrow. Under microaerophilic conditions as oxygen becomes limiting, cydAB is optimally active and ArcA may successfully compete Fnr to activate the regulator under these conditions (Bunn and Poyton, 1996).

View larger version (118K): [in a new window]   Fig. 6. Example of Vendian flossils from the Ediacara Hills region in Southeast Australia. Arrows indicate microorganisms with amoeboid, hydra and sponge-like features, many with cilia-like structures indicating motility. (From the University of California at Berkeley Paleontological Museum: with permission).

View larger version (10K): [in a new window]   Fig. 7. Positive and negative regulation of gene expression in yeast. Under anaerobic conditions the LORE-BP (low oxygen response element binding protein) is activated. The mechanism of activation parallels that of higher eukaryotes insofar as anaerobic induction can be mimicked by transition metals and desferroximine. The possible involvement of a proline-hydroxyproline active site has not been demonstrated in this system as it has in the HIF-1 pathway (see Fig. 9). LORE-BP is a positive-acting transcription factor that binds and activates hypoxia-response genes with the sequence ACTCAACAA. The HAP1-Rox-1 pathway operates in parallel with the LORE pathway. HAP-1 requires heme as a cofactor and is activated under aerobic conditions when heme levels are high. HAP-1 is a positive transcription factor for multiple aerobic genes (genes required for aerobic metabolism and functions) through binding to the recognition sequence CGG(N6)CGG. The ROX-1 promoter region contains the HAP-1 binding site and is activated by HAP-1. ROX-1 is a repressor that negatively regulates (hypoxic) genes containing the sequence CCATTGTTCTC. Consequently HAP-1 coordinately regulates both aerobic and hypoxic genes.

View larger version (79K): [in a new window]   Fig. 8. (A) The Rhynie valley in Scotland. (B) Devonian fossilized fern leaves about 0.8 billion years old. (From the University of California at Berkeley Paleontological Museum; with permission).

View larger version (19K): [in a new window]   Fig. 9. Regulation of mammalian glycolytic enzyme genes by the HIF-1 and Sp1 family transcription factors. A proline residue at position 564 on the HIF-1 protein (illustrated as a helix-turn-helix structure) is hydroxylated at physiological oxygen tension, causing a conformational change and rendering the protein susceptible to ubiquitination (Ub). Ubiquitinated protein is rapidly degraded by the proteosome. Under hypoxia, this pathway is blocked; HIF-1 accumulates, dimerizes the aryl hydrocarbon nuclear transporter (ARNT), translocates to the nucleus, and activates responsive genes including glycolytic enzymes by binding to sequences containing the consensus ACGT site. In a second redox-regulated step, p300 can only be recruited to activate the HIF-1 complex when the Asp-851 residue is not hydroxylated. In a parallel hypoxia-regulated pathway, Sp1 and Sp3 (zinc fingers) compete for binding to GC-rich DNA sequences; Sp1 is a positive transcriptional activator while Sp3 can repress transcription. Hypoxia favors degradation of Sp3 (by an unknown mechanism), promoting the full inducing activity of Sp1.