Structural insights into Pot1-ssDNA, Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex.

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.

Transcriptional regulation of oncogenic protein kinase Cϵ (PKCϵ) by STAT1 and Sp1 proteins.

Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼ 1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and -105 bp (region A) and -921 and -796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and -269/-247 as well as STAT1 sites in positions -880/-869 and -793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics.

Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity.

Helicobacter pylori cagA-positive strains are associated with gastritis, ulcerations and gastric adenocarcinoma. CagA is delivered into gastric epithelial cells and, on tyrosine phosphorylation, specifically binds and activates the SHP2 oncoprotein, thereby inducing the formation of an elongated cell shape known as the 'hummingbird' phenotype. In polarized epithelial cells, CagA also disrupts the tight junction and causes loss of apical-basolateral polarity. We show here that H. pylori CagA specifically interacts with PAR1/MARK kinase, which has an essential role in epithelial cell polarity. Association of CagA inhibits PAR1 kinase activity and prevents atypical protein kinase C (aPKC)-mediated PAR1 phosphorylation, which dissociates PAR1 from the membrane, collectively causing junctional and polarity defects. Because of the multimeric nature of PAR1 (ref. 14), PAR1 also promotes CagA multimerization, which stabilizes the CagA-SHP2 interaction. Furthermore, induction of the hummingbird phenotype by CagA-activated SHP2 requires simultaneous inhibition of PAR1 kinase activity by CagA. Thus, the CagA-PAR1 interaction not only elicits the junctional and polarity defects but also promotes the morphogenetic activity of CagA. Our findings revealed that PAR1 is a key target of H. pylori CagA in the disorganization of gastric epithelial architecture underlying mucosal damage, inflammation and carcinogenesis.

Role of partitioning-defective 1/microtubule affinity-regulating kinases in the morphogenetic activity of Helicobacter pylori CagA.

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d >or= PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.

Structural and functional diversity in the PAR1b/MARK2-binding region of Helicobacter pylori CagA.

Helicobacter pylori (H. pylori) cagA-positive strains are associated with gastritis, peptic ulcerations, and gastric adenocarcinoma. Upon delivery into gastric epithelial cells, the cagA-encoded CagA protein specifically binds and aberrantly activates SHP-2 oncoprotein in a manner that is dependent on CagA tyrosine phosphorylation. CagA-deregulated SHP-2 then elicits aberrant Erk activation while causing an elongated cell shape known as the hummingbird phenotype. In polarized epithelial cells, CagA also binds to PAR1b/MARK2 and inhibits the PAR1b kinase activity, thereby disrupting tight junctions and epithelial cell polarity independent of CagA tyrosine phosphorylation. We show here that the CagA-multimerization (CM) sequence that mediates interaction of CagA with PAR1b is not only essential for the CagA-triggered junctional defects but also plays an important role in induction of the hummingbird phenotype by potentiating CagA-SHP-2 complex formation. We also show that the CM sequence of CagA isolated from East Asian H. pylori (referred to as the E-CM sequence) binds PAR1b more strongly than that of CagA isolated from Western H. pylori (referred to as the W-CM sequence). Within Western CagA species, the ability to bind PAR1b is proportional to the number of W-CM sequences. Furthermore, the level of PAR1b-binding activity of CagA correlates with the magnitude of junctional defects and the degree of hummingbird phenotype induction. Our findings reveal that structural diversity in the CM sequence is an important determinant for the degree of virulence of CagA, a bacterial oncoprotein that is associated with gastric carcinogenesis.

Evaluating the Association of Eight Polymorphisms with Cancer Susceptibility in a Han Chinese Population.

BACKGROUND: The identification of susceptibility genes for specific types of cancer can provide necessary information for the complete characterization of cancer syndromes. Eight single nucleotide polymorphisms (SNPs), rs465498, rs17728461, rs4488809, rs753955, rs13361707, rs9841504, rs2274223, and rs13042395, were reported by genome wide association studies (GWASs) to be closely related to the susceptibility of lung cancer (LC), gastric cancer (GC) or esophageal cancer (EC) in Han population from northern or southern China. However, Chinese Han people from different geographic areas may have different genetic backgrounds. This study aims to assess the genetic associations of the eight SNPs mentioned above with three cancers risk in a Han population from northwest China. METHODS: A total of 186 cancer-free controls and 436 cases with non-small cell lung cancer (NSCLC) (159 cases), non-cardia GC (167 cases) or EC (110 cases) were enrolled in this study. Chi-square test and polytomous logistic regression analyses were used to estimate the association between eight cancer-related SNPs and three cancers in a Han Chinese population from northwest China. The logistic regression results were adjusted for confounding factors and Benjamini and Hochberg False Discovery Rate (FDR) method was used to adjust the multiple hypothesis tests. Association analyses by cigarette smoking or alcohol drinking status were analyzed by crossover analyses. RESULTS: One of the eight SNPs, rs17728461 was associated with NSCLC susceptibility (in a heterozygous model, OR = 0.44, 95% CI = 0.27-0.72, p = 0.001). Two SNPs, rs753955 and rs13042395, were associated with the risk of non-cardia GC in different genetic models (p < 0.05). No SNPs were associated with EC. The crossover analyses showed that the rs13042395 CT genotype, combined with cigarette smoking or alcohol drinking, could further increase the risk for non-cardia GC (p < 0.05). CONCLUSIONS: These results indicated that rs17728461 may be specifically associated with the risk of NSCLC. rs753955 and rs13042395 were specifically associated with susceptibility to non-cardia GC in Ningxia Han Chinese. Susceptibility-associated polymorphisms in the northwestern Han Chinese were not very consistent with those in the northern Han Chinese or southern Han Chinese. The validation of these findings with a functional evaluation and a larger population is still required.

Intercellular adhesion molecule 1/2 and E-selectin in plasma cell mastitis: immunohistochemical study of 35 cases.

Plasma cell mastitis (PCM) is one of the most frequently encountered inflammatory diseases of the nonlactating breast. Histologically, PCM is characterized by infiltration of relatively abundant plasma cells into the mammary ducts. Its pathogenesis has remained unknown. In this study, we immunolocalized intercellular adhesion molecule (ICAM) 1 and 2 and E-selectin, all of which play pivotal roles in the inflammatory process, in 35 cases of PCM. We then compared the results with those of non-PCM and nonpathologic breast tissue. In the ductal epithelium, ICAM-1 immunoreactivity was significantly more pronounced in PCM than in non-PCM (P = .045). Both ICAM-1 (P < .001) and ICAM-2 (P = .001) were significantly more pronounced in PCM than in nonpathologic breast tissue. However, no significant differences in ICAM-2 and E-selectin immunoreactivity were detected between ductal epithelium of PCM and non-PCM. ICAM-1, but not ICAM-2 or E-selectin, demonstrated significantly higher immunoreactivity in endothelial cells of PCM than in nonpathologic breast (P < .001). These results all suggest that ICAM-1 in both ductal epithelium and endothelium plays important roles in the inflammatory process of PCM, possibly through margination, extravasation, and attachment of plasma cells and lymphocytes, which may result in continuous inflammatory cell homing to ductal epithelial cells.

Structural basis and functional consequence of Helicobacter pylori CagA multimerization in cells.

Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDLSKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosinephosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.