cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1␣ stabilization during hypoxia.Hypoxia initiates transcription of a number of gene products that help to sustain the supply of O 2 to tissues and to enhance cell survival during severe O 2 deprivation. Gene products that augment O 2 supply at the tissue level include erythropoietin (Epo) 1 which increases the proliferation of erythrocytes, tyrosine hydroxylase which is necessary for the synthesis of the neurotransmitter dopamine in the carotid bodies, and the angiogenic factor VEGF which stimulates growth of new capillaries (1-3). At the cellular level, gene products that enhance survival during hypoxia include the glycolytic enzymes and the glucose transporters Glut1 and Glut3 (4). The induction of these genes is mediated by hypoxia-inducible factor-1 (HIF-1) (5-7), a heterodimeric transcription factor consisting of HIF-1␣ and the aryl hydrocarbon nuclear translocator (ARNT or HIF-1␤) subunits (7-9). The significance of HIF-1 in transcriptional regulation was recently demonstrated by the marked decrease in mRNA expression of VEGF and glycolytic enzymes seen during hypoxia in HIF-1␣-or ARNT-deficient murine embryonic stem cells (10 -12).The mechanism by which HIF-1 activation is initiated during hypoxia remains unclear. Both HIF-1␣ and ARNT mRNAs are constitutively expressed, indicating that functional activity of the HIF-1␣⅐ARNT complex is regulated by post-transcriptional events. ARNT levels are not significantly affected by [O 2 ], whereas HIF-1␣ protein is rapidly degraded under normoxic conditions by the ubiquitin-proteasome system (13,14). Hypoxia enhances HIF-1␣ protein levels by inhibiting its degradation, thereby allowing it to accumulate, to dimerize with ARNT, and to bind to the hypoxia-responsive element (HRE) in the promoter or enhancer regions of various genes. Thus, the functional HIF-1␣⅐ARNT complex is primarily regulated by the abundance of the HIF-1␣ subunit.Although much has been learned about the role of HIF-1 in controlling the expression of hypoxia-responsive genes, the underlying mechanism by which cells detect the decrease in [O 2 ] and initiate the stabilization of HIF-1␣ is not known. Presently, four diverse O 2 -sensing mechanisms have been proposed to mediate the transcriptional response to hypoxia (15). Two of these models postulate the involvement of an iron-containing unit in the form of either a heme group or an iron/sulfur cluster, which undergoes a change in activity during hypoxia that triggers the transcriptional response. These models are supported by the observation that cobaltous ions, or alternatively the iron chelator desferrioxamine (DFO), stabilize HIF-1␣ under normoxic conditions (16). However, no specific proteins with this role have been identified in mammalian systems. Two other models involve the generation of reactive oxygen species (ROS) by a f...

BRCA1, a multi-domain protein, is mutated in a large percentage of hereditary breast and ovarian cancers. BRCA1 is most often mutated in three domains or regions: the N-terminal RING domain, exons 11–13, and the BRCT domain. The BRCA1 RING domain is responsible for the E3 ubiquitin ligase activity of BRCA1 and mediates interactions between BRCA1 and other proteins. BRCA1 ubiquitinates several proteins with various functions. The BRCA1 BRCT domain binds to phosphoproteins with specific sequences recognized by both BRCA1 and ATM/ATR kinases. Structural studies of the RING and BRCT domains have revealed the molecular basis by which cancer causing mutations impact the functions of BRCA1. While no structural data is available for the amino acids encoded by exons 11–13, multiple binding sites and functional domains exist in this region. Many mutations in exons 11–13 have deleterious effects on the function of these domains. In this mini-review, we examine the structure-function relationships of the BRCA1 protein and the relevance to cancer progression.

No abstract

Manganese-superoxide dismutase (Sod2) removes mitochondrially derived superoxide (O 2 . ) at near-diffusion limiting rates and is the only antioxidant enzyme whose expression is regulated by numerous stimuli. Here it is shown that Sod2 also serves as a source of the intracellular signaling molecule H 2 O 2 . Sod2-dependent increases in the steady-state levels of H 2 O 2 led to ERK1/2 activation and subsequent downstream transcriptional increases in matrix metalloproteinase-1 (MMP-1) expression, which were reversed by expression of the H 2 O 2 -detoxifying enzyme, catalase. In addition, a single nucleotide polymorphism has recently been identified (1G/2G) at base pair ؊1607 that creates an Ets site adjacent to an AP-1 site at base pair ؊1602 and has been shown to dramatically enhance transcription of the MMP-1 promoter. Luciferase promoter constructs containing either the 1G or 2G variation were 25-or 1000-fold more active when transiently transfected into Sod2-overexpressing cell lines, respectively. The levels of MMP-2, -3, and -7 were also increased in the Sod2-overexpressing cell lines, suggesting that Sod2 may function as a "global" redox regulator of MMP expression. In addition, Sod2 ؊/؉ mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study provides evidence that the modulation of Sod2 activity by a wide array of pathogenic and inflammatory stimuli may be utilized by the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.

Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the ␣-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the ⌬bssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.