H(+)-translocating pyrophosphatases (H(+)-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PP(i)) hydrolysis. H(+)-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H(+)-PPases are unclear. Here we report the crystal structure of a Vigna radiata H(+)-PPase (VrH(+)-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35 Å resolution. Each VrH(+)-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg(2+) ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PP(i) hydrolysis, and H(+) is then transported into the vacuolar lumen through a pathway consisting of Arg 242, Asp 294, Lys 742 and Glu 301. We propose a working model of the mechanism for the coupling between proton pumping and PP(i) hydrolysis by H(+)-PPases.
SUMMARY Biomarkers for predicting prognosis are critical to treating colorectal cancer (CRC) patients. We found that CSN6, a subunit of COP9 signalosome, is overexpressed in CRC samples and that CSN6 overexpression is correlated with poor patient survival. Mechanistic studies revealed that CSN6 is deregulated by EGFR signaling, in which ERK2 binds directly to CSN6 Leu163/Val165 and phosphorylates CSN6 at Ser148. Furthermore, CSN6 regulated β-Trcp and stabilizes β-catenin expression by blocking the ubiquitin-proteasome pathway, thereby promoting CRC development. High CSN6 expression was positively correlated with ERK2 activation and β-catenin overexpression in CRC samples, and inhibiting CSN6 stability with cetuximab reduced colon cancer growth. Taken together, our study’s findings indicate that the deregulation of β-catenin by ERK2-activated CSN6 is important for CRC development.
Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. Leptospirosis is the most widespread zoonosis, particularly in warm and humid climates (1-3). The clinical manifestations of leptospirosis occur when humans acquire the pathogen Leptospira from animals (4) via skin or gastrointestinal contact with water, food, or soil that has been contaminated with urine from infected animals. Clinical symptoms of leptospirosis vary from mild to severe, and potentially lethal forms are characterized by high fever, bleeding, and renal failure (5). The kidney is the main target of Leptospira in both acute and chronic infections (1, 6). Acute kidney injury as a result of tubulointerstitial nephritis is an early and primary manifestation of systemic leptospirosis (6). In chronic infections, Leptospira may colonize and persist in the proximal tubule, leading to carrier status, and thus may lead to chronic tubulointerstitial nephritis and fibrosis (1).Outer membrane proteins (OMPs) 2 and lipopolysaccharide located on the outer membrane are major antigens that confer immunity to Leptospira (7). Leptospiral OMPs are likely to have roles in host-pathogen interactions (7,8). They elicit inflammation and lead to tubular injury through Tolllike receptor-dependent pathways. Subsequently, the nuclear transcription factor NF-B and the mitogen-activated protein kinases are activated, leading to the differential induction of chemokines and cytokines relevant to tubular inflammation (9 -11).Bacterial pathogenesis can occur when mammalian extracellular matrix (ECM) molecules interact with bacterial cell surface proteins (12). These interactions can be used by the bacterium to adhere to tissues, evade an immune response, and enter the host. ECM components in mammalian cells are diverse and include fetuin, laminin-1, collagen, and plasma fibronectin. Leptospira recognizes ECM molecules and adheres to the host cell though pathogenic OMPs such as immunoglobulin-like protein (13), endostatin-like protein (14), and the lipoprotein LipL32 (10).LipL32 is a major OMP in Leptospira and is highly conserved among pathogenic species. It is expressed during mammalian infection by Leptospira (7). LipL32 is a virulent microbial surface component that recognizes adhesive matrix molecules (15). LipL32 induces inflammation via NF-B and mitogenactivated protein kinase pathways (4) through TLR2 (10). LipL32 adheres to the host cell by recognizing ECM molecules such as type IV collagen and plasma fibronectin (F30 and F45) (15, 16). The heparin-binding domain of F30 and the gelatinbinding domain of F45 are involved in these host cell interactions (16). Although LipL32 has been suggested to have a role in pathogenicity, a recent report indicates that LipL32 is not essential for survival or virulence (17). Thus, the role of LipL...
GA (glucoamylase) hydrolyses starch and polysaccharides to beta-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with beta-cyclodextrin (SBD-betaCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp(47)) and II (Tyr(32)), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr(32) was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.
Biomarkers for predicting chemotherapy response are important to treatment of colorectal cancer (CRC) patients. Cryptochrome 2 (CRY2) is a circadian clock protein involved in cell cycle, but the biological consequences of this activity in cancer are poorly understood. We set up biochemical and cell biology analyses to analyze CRY2 expression and chemoresistance. Here we report that CRY2 is overexpressed in chemoresistant CRC samples, and CRY2 overexpression is correlated with poor patient survival. Knockdown CRY2 increased colorectal cancer sensitivity to oxaliplatin in colorectal cancer cell. We also identify FBXW7 as a novel E3 ubiquitin ligase for targeting CRY2 through proteasomal degradation. Mechanistic studies show that CRY2 is regulated by FBXW7, in which FBXW7 binds directly to phosphorylated Thr300 of CRY2. Furthermore, FBXW7 expression leads to degradation of CRY2 through enhancing CRY2 ubiquitination and accelerating CRY2’s turnover rate. High expressed FBXW7 downregulates CRY2 and increases colorectal cancer cells sensitivity to chemotherapy. Low FBXW7 expression is correlated with high CRY2 expression in CRC patient samples. Also, low FBXW7 expression is correlated with poor patient survival. Taken together, our findings indicate that the upregulation of CRY2 caused by downregulation of FBXW7 may be a novel prognostic biomarker and may represent a new therapeutic target in colorectal cancer.
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