High GILT Expression and an Active and Intact MHC Class II Antigen Presentation Pathway Are Associated with Improved Survival in Melanoma.

The MHC class I Ag presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of tumors. However, the clinical significance of the MHC class II Ag presentation pathway in melanoma cells is less clear. In Ag-presenting cells, IFN-γ-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma Ags. Although not expressed in benign melanocytes of nevi, GILT and MHC class II expression is induced in malignant melanocytes in a portion of melanoma specimens. Analysis of The Cancer Genome Atlas cutaneous melanoma data set showed that high GILT mRNA expression was associated with improved overall survival. Expression of IFN-γ, TNF-α, and IL-1ß was positively associated with GILT expression in melanoma specimens. These cytokines were capable of inducing GILT expression in human melanoma cells in vitro. GILT protein expression in melanocytes was induced in halo nevi, which are nevi undergoing immune-mediated regression, and is consistent with the association of GILT expression with improved survival in melanoma. To explore potential mechanisms of GILT's association with patient outcome, we investigated pathways related to GILT function and expression. In contrast to healthy skin specimens, in which the MHC class II pathway was nearly uniformly expressed and intact, there was substantial variation in the MHC class II pathway in the The Cancer Genome Atlas melanoma specimens. Both an active and intact MHC class II pathway were associated with improved overall survival in melanoma. These studies support a role for GILT and the MHC class II Ag presentation pathway in melanoma outcome.

Purification, characterization and crystallization of menaquinol:fumarate oxidoreductase from the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

The integral membrane protein complex, menaquinol:fumarate oxidoreductase (mQFR) has been purified, identified and characterized from the thermophilic green filamentous anoxygenic photosynthetic bacterium Chloroflexus aurantiacus. The complex is composed of three subunits: a 74 kDa flavoprotein that contains a covalently bound flavin adenine dinucleotide, a 28 kDa iron-sulfur cluster-containing polypeptide, and a 27 kDa transmembrane polypeptide, which is also the binding site of two b-type hemes and two menaquinones. The purified complex has an apparent molecular mass of 260 kDa by blue-native PAGE, which is indicative of a native homodimeric form. The isolated complex is active in vitro in both fumarate reduction and succinate oxidation. It has been analyzed by visible absorption, redox titration, chemical analysis and EPR spectroscopy. In addition, phylogenetic analysis shows that the QFR of both C. aurantiacus and Chlorobium tepidum are most closely related to those found in the delta-proteobacteria. The purified enzyme was crystallized and X-ray diffraction data obtained up to 3.2 A resolution.

Metabolic flexibility revealed in the genome of the cyst-forming alpha-1 proteobacterium Rhodospirillum centenum.

BACKGROUND: Rhodospirillum centenum is a photosynthetic non-sulfur purple bacterium that favors growth in an anoxygenic, photosynthetic N2-fixing environment. It is emerging as a genetically amenable model organism for molecular genetic analysis of cyst formation, photosynthesis, phototaxis, and cellular development. Here, we present an analysis of the genome of this bacterium. RESULTS: R. centenum contains a singular circular chromosome of 4,355,548 base pairs in size harboring 4,105 genes. It has an intact Calvin cycle with two forms of Rubisco, as well as a gene encoding phosphoenolpyruvate carboxylase (PEPC) for mixotrophic CO2 fixation. This dual carbon-fixation system may be required for regulating internal carbon flux to facilitate bacterial nitrogen assimilation. Enzymatic reactions associated with arsenate and mercuric detoxification are rare or unique compared to other purple bacteria. Among numerous newly identified signal transduction proteins, of particular interest is a putative bacteriophytochrome that is phylogenetically distinct from a previously characterized R. centenum phytochrome, Ppr. Genes encoding proteins involved in chemotaxis as well as a sophisticated dual flagellar system have also been mapped. CONCLUSIONS: Remarkable metabolic versatility and a superior capability for photoautotrophic carbon assimilation is evident in R. centenum.

Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations, and pH.

The effects of Cl(-), Mn(2+), Ca(2+), and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl(-), and strongly inhibited above this concentration. This enzyme is activated by Mn(2+) and stimulated somewhat by Ca(2+). The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl(-) up to 0.4 M. Ca(2+) appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn(4) complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.

Hypothesis: the peroxydicarbonic acid cycle in photosynthetic oxygen evolution.

Peroxydicarbonic acid (Podca), a proposed intermediate in photosynthetic oxygen evolution, was synthesized electrochemically. Consistent with literature descriptions of this compound, it was shown to be a highly reactive molecule, spontaneously hydrolyzed to H2O2, as well as susceptible to oxidative and reductive decomposition. In the presence of Mn2+ or Co2+, Podca was quickly broken down with release of O2. The liberation of O2, however, was partially suppressed at high O2 concentrations. In the presence of Ca-washed photosystem II-enriched membranes lacking extrinsic proteins, Podca was decomposed with the release of O2, but only under conditions favoring photosynthetic electron flow (light plus a Hill oxidant). A model is proposed that details how peroxydicarbonic acid could act as an oxygen-evolving intermediate. The hypothesis is consistent with the well-established Kok model and with recent findings related to the chemistry of oxygen evolution.

Carbonic anhydrase activity of the photosystem II OEC33 protein from pea.

The purpose of this study was to identify the location of one of the two sources of carbonic anhydrase (CA) activity associated with the PSII complex in chloroplast membranes. We tested the hypothesis that the extrinsic 33 kDa protein, OEC33, associated with the oxygen-evolving complex (OEC), is one source of CA activity. We found that precursor OEC33 expressed in Escherichia coli exhibits CA activity, but the expressed precursors of OEC24 or OEC17 do not. The CA activity of OEC33 remained after treatment at 90 degrees C for 15 min. Additional biochemical evidence supports the hypothesis. Only those wash treatments that remove the OEC33 from PSII also remove CA activity. Both immunoblot and CA activity show that the CA tracks the OEC33, in parallel, when PSII undergoes washing at different CaCl2 concentrations. The OEC33 protein purified by HiTrap Q anion exchange chromatography has CA activity that is inhibited by an antibody against OEC33. PSII membranes washed with 1 M CaCl2 to remove OEC33 can be reconstituted either with extracted, purified, OEC33 or with the E. coli-expressed precursor OEC33. Reconstitution partially restores both oxygen evolution and CA activity. For maximal CA activity, OEC33 requires manganese as a cofactor.

Extrinsic photosystem II carbonic anhydrase in maize mesophyll chloroplasts.

One form of carbonic anhydrase (CA) has been observed in maize (Zea mays) thylakoids and photosystem II (PSII)-enriched membranes. Here, we show that an antibody produced against a thylakoid lumen-targeted CA found in Chlamydomonas reinhardtii reacts with a single 33-kD polypeptide in maize thylakoids. With immunoblot analysis, we found that this single polypeptide could be identified only in mesophyll thylakoids and derived PSII membranes, but not in bundle sheath thylakoids. Likewise, a CA activity assay confirmed a large amount of activity in mesophyll, but not in bundle sheath membranes. Immunoblot analysis and CA activity assay showed that the maximum CA can be obtained in the supernatant of the PSII-enriched membranes washed with 1 M CaCl(2), the same procedure used to remove all extrinsic lumenal proteins from PSII. Because this CA reacts with an antibody to lumen-directed CA in C. reinhardtii, and because it can be removed with 1 M CaCl(2) wash, we refer to it tentatively as extrinsic CA. This is to distinguish it from another form of CA activity tightly bound to PSII membranes that remains after CaCl(2) wash, which has been described previously. The function of extrinsic CA is not clear. It is unlikely to have the same function as the cytoplasmic CA, which has been proposed to increase the HCO(-)(3) concentration for phosphoenolpyruvate carboxylase and the C(4) pathway. We suggest that because the extrinsic CA is associated only with thylakoids doing linear electron flow, it could function to produce the CO(2) or HCO(-)(3) needed for PSII activity.