The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. Hydroxylation of one or two critical prolines by specific hydroxylases (P4Hs) targets their HIF-␣ subunits for proteasomal degradation. By studying the three human HIF-P4Hs, we found that the longest and shortest isoenzymes have major transcripts encoding inactive polypeptides, which suggest novel regulation by alternative splicing. Recombinant HIFP4Hs expressed in insect cells required peptides of more than 8 residues, distinct differences being found between isoenzymes.

The collagen superfamily of proteins now contains at least 19 proteins formally defined as collagens and an additional ten proteins that have collagen-like domains. The most abundant collagens form extracellular fibrils or network-like structures, but the others fulfill a variety of biological functions. Some of the eight highly specific post-translational enzymes involved in collagen biosynthesis have recently been cloned. Over 400 mutations in 6 different collagens cause a variety of human diseases that include osteogenesis imperfecta, chondrodysplasias, some forms of osteoporosis, some forms of osteoarthritis, and the renal disease known as the Alport syndrome. Many of the disease phenotypes have been produced in transgenic mice with mutated collagen genes. There has been increasing interest in the possibility that the unique post-translational enzymes involved in collagen biosynthesis offer attractive targets for specifically inhibiting excessive fibrotic reactions in a number of diseases. A number of experiments suggest it may be possible to inhibit collagen synthesis with oligo-nucleotides or antisense genes.

The collagen superfamily of proteins plays a dominant role in maintaining the integrity of various tissues and also has a number of other important functions. The superfamily now includes more than 20 collagen types with altogether at least 38 distinct polypeptide chains, and more than 15 additional proteins that have collagen-like domains. Most collagens form polymeric assemblies, such as fibrils, networks and filaments, and the superfamily can be divided into several families based on these assemblies and other features. All collagens also contain noncollagenous domains, and many of these have important functions that are distinct from those of the collagen domains. Major interest has been focused on endostatin, a fragment released from type XVIII collagen, which potently inhibits angiogenesis and tumour growth. Collagen synthesis requires eight specific post-translational enzymes, some of which are attractive targets for the development of drugs to inhibit collagen accumulation in fibrotic diseases. The critical roles of collagens have been clearly illustrated by the wide spectrum of diseases caused by the more than 1,000 mutations that have thus far been identified in 22 genes for 12 out of the more than 20 collagen types. These diseases include osteogenesis imperfecta, many chondrodysplasias, several subtypes of the Ehlers-Danlos syndrome, Alport syndrome, Bethlem myopathy, certain subtypes of epidermolysis bullosa, Knobloch syndrome and also some cases of osteoporosis, arterial aneurysms, osteoarthrosis, and intervertebral disc disease. The characterization of mutations in additional collagen genes will probably add further diseases to this list. Mice with genetically engineered collagen mutations have proved valuable for defining the functions of various collagens and for studying many aspects of the related diseases.

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The activity of hypoxia-inducible transcription factor HIF, an ␣␤ heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-␣ subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1␣ sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2␣ peptides was far less efficient than that of the corresponding HIF-1␣ peptides. The K m of FIH for O 2 was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For example, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response.The hypoxia-inducible transcription factor HIF 1 has a major role in the conserved oxygen-sensitive response pathway that is activated in hypoxic cells. HIF-regulated hypoxia-inducible genes are involved in angiogenesis, vascularization, and anaerobic energy production, for instance. HIFs are ␣␤ heterodimers in which the stability of the ␣ subunit is regulated in an oxygen-dependent manner (for reviews, see Refs. 1-4). Hydroxylation of at least one of two proline residues, Pro 402 and Pro 564 , in the oxygen-dependent degradation domain of human HIF-1␣ mediates the interaction of HIF-␣ with the von Hippel Lindau E3 (ubiquitin-protein isopeptide ligase) ubiquitin ligase complex that targets it for rapid proteasomal degradation under normoxic conditions (5-8). This hydroxylation is catalyzed in humans by three novel cytoplasmic and nuclear HIF prolyl 4-hydroxylases (9 -11) that are distinct from the well characterized collagen prolyl 4-hydroxylases, which reside in the lumen of the endoplasmic reticulum (12-16). Under hypoxic conditions, the oxygen-requiring process of hydroxylation is prevented, and HIF-␣ escapes degradation and dimerizes with HIF-␤, the dimer then recognizing a specific element in the promoters of hypoxia-responsive target genes (1-4).Transcriptional activation in an oxygen-dependent manner is another key step that regulates HIF-␣ activity. Hydroxylation of a specific asparagine residue, Asn 80...