Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the ␣-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical ␣-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the threedimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO 2 hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated ␣-CAs, further prospects for the rational drug design of isozymespecific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 3 tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 3 aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Coimmunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds ␤ 2 -microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.Hereditary hemochromatosis (HH) 1 is an autosomal recessive disorder of iron metabolism and represents one of the most common inherited disorders in individuals of Northern European descent with an estimated carrier frequency between 1 in 8 and 1 in 10 (1, (2). In patients with HH, excessive iron deposition in a variety of organs leads to multi-organ dysfunction. Recently, we reported a mutation in a novel MHC class I-like gene, called HLA-H (3). Eighty-three percent of HH patient DNAs were found to be homozygous for this mutation, which consists of a single base transition of G to A and results in a change of cysteine 282 3 tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4 -6), providing further support that HLA-H is the primary HH locus. A second missense mutation, histidine 63 3 aspartate (H63D), was also reported that was enriched in heterozygotes with the C282Y mutation (eight of nine cases) (3). The specific role that either of these mutations in HLA-H play in the etiology of HH disease has not been elucidated.The HLA-H protein is similar to MHC class I family molecules including HLA-A2, nonclassical class I molecules such as HLA-G, and the human neonatal Fc receptor (FcRn). All four of the invariant cysteine residues that form disulfide bridges in the ␣ 2 and ␣ 3 domains of MHC class I family members are present in the HLA-H protein. One of these conserved cysteine residues is altered in the C282Y mutation. The integrity of the conserved disulfide linkages has been suggested to be critical for proper maintenance of the secondary and tertiary structure of the protein allowing interactions with accessory molecules such as ␤ 2 -microglobulin (7). Importantly, the functional significance of an interaction between ␤ 2 -microglobulin and an unknown class I-like molecule in HH disease was s...

Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 ؎ 5% vs. 77 ؎ 3%, P < 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 ؎ 450 vs. 255 ؎ 23 g͞g dry wt, P < 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload.

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To determine if alcoholic liver fibrogenesis is exacerbated by dietary iron supplementation, carbonyl iron (0.25% wt/ vol) was intragastrically infused with or without ethanol to rats for 16 wk. Carbonyl iron had no effect on blood alcohol concentration, hepatic biochemical measurements, or liver histology in control animals. In both ethanol-fed and control rats, the supplementation produced a two-to threefold increase in the mean hepatic non-heme iron concentration but it remained within or near the range found in normal human subjects. As previously shown, the concentrations of liver malondialdehyde (MDA),' liver 4-hydroxynonenal (4HNE), and serum aminotransferases (ALT, AST) were significantly elevated by ethanol infusion alone. The addition of iron supplementation to ethanol resulted in a further twofold increment in mean MDA, 4HNE, ALT, and AST. On histological examination, focal fibrosis was found < 30% of the rats fed ethanol alone. In animals given both ethanol and iron, fibrosis was present in all, with a diffuse centralcentral bridging pattern in 60%, and two animals (17%) developed micronodular cirrhosis. The iron-potentiated alcoholic liver fibrogenesis was closely associated with intense and diffuse immunostaining for MDA and 4HNE adduct epitopes in the livers. Furthermore, in these animals, accentuated increases in procollagen al (I) and TGF#61 mRNA levels were found in both liver tissues and freshly isolated hepatic stellate cells, perisinusoidal cells believed to be a major source of extracellular matrices in liver fibrosis. The dietary iron supplementation to intragastric ethanol infusion exacerbates hepatocyte damage, promotes liver fibrogenesis, and produces evident cirrhosis in some animals.