A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.

Activation of YAP, a Hippo pathway effector, is an important resistance mechanism to BRAF inhibitor (BRAFi) in melanoma. Emerging evidence also suggests that YAP is involved in suppression of the antitumor immune response. However, the potential direct impact of YAP activity on cytotoxic T-cell immune responses has not been explored yet. Here, we show that BRAFi-resistant melanoma cells evade CD8 T-cell immune responses in a PD-L1-dependent manner by activating YAP, which synchronously supports melanoma cell survival upon BRAF inhibition. PD-L1 expression is elevated in BRAFi-resistant melanoma cells, in which YAP is robustly activated, and YAP knockdown decreases PD-L1 expression. In addition, constitutively active YAP (YAP-5SA) increases PD-L1 expression by binding to an upstream enhancer of the PD-L1 gene and potentiating its transcription. Both BRAFi-resistant and YAP-5SA-expressing melanoma cells suppress the cytotoxic function and cytokine production of Melan-A-specific CD8 T cells, whereas anti-PD-1 antibody reverses the YAP-mediated T-cell suppression. Moreover, nuclear enrichment of YAP in clinical melanoma samples correlates with increased PD-L1 expression. Our findings show that YAP directly mediates evasion of cytotoxic T-cell immune responses in BRAFi-resistant melanoma cells by upregulating PD-L1, and targeting of YAP-mediated immune evasion may improve prognosis of melanoma patients.

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni 2؉ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent K m values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V max values (at 90°C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k cat /K m ) of the enzyme were 74.8 mM ؊1⅐ min ؊1 (L-arabinose) and 8.5 mM ؊1 ⅐ min ؊1 (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn 2؉ and/or Co 2؉ than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80°C.Of the eubacteria whose genomes have been sequenced to date, Thermotoga maritima has the highest percentage of genes that are most similar to archaeal genes (20). This bacterium is also of evolutionary importance, because small-subunit rRNA phylogeny has shown that it is one of the deepest and most slowly evolving lineages of the eubacteria. Thus, it could be useful for elucidating the evolutionary relationship between thermophilic eubacteria and archaea. In addition, it metabolizes many simple and complex carbohydrates, including glucose, sucrose, starch, cellulose, and xylan (9). Thermostable enzymes from T. maritima involved in carbohydrate metabolism are very attractive for industrial applications (4,15,24).Almost 7% of the predicted coding sequences in the T. maritima genome are involved in metabolism of simple and complex sugars (20). Several genes encode proteins involved in arabinose metabolism. Among them, the araA gene encoding L-arabinose isomerase (AI) (EC 5.3.1.4), which catalyzes the conversion of L-arabinose to L-ribulose, not only is important for pentose sugar isomerization in vivo but also is very attractive for use in the bioconversion of D-galactose into D-tagatose in vitro (5, 25) (Fig. 1).The ketohexose D-tagatose has a sweetness value (92%) equivalent to that of sucrose but is poorly digested (18,32). This compound has been found to be a safe low-calorie sweetener in food products and is classified as a generally recognized as safe substance in the United States. It has also been classified as generally recognized as safe for u...

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