Crystal structure of the Propionibacterium acnes surface sialidase, a drug target for P. acnes-associated diseases.

Propionibacterium acnes, though generally considered part of the normal flora of human skin, is an opportunistic pathogen associated with acne vulgaris as well as other diseases, including endocarditis, endophthalmitis and prosthetic joint infections. Its virulence potential is also supported by knowledge gained from its sequenced genome. Indeed, a vaccine targeting a putative cell wall-anchored P. acnes sialidase has been shown to suppress cytotoxicity and pro-inflammatory cytokine release induced by the organism, and is proposed as an alternative treatment for P. acnes-associated diseases. Here, we report the crystal structures of the surface sialidase and its complex with the transition-state mimic Neu5Ac2en. Our structural and kinetic analyses, together with insight from a glycan array screen, which probes subtle specificities of the sialidase for α-2,3-sialosides, provide a basis for the structure-based design of novel small-molecule therapeutics against P. acnes infections.

Inhibition of nucleophosmin 1 suppresses colorectal cancer tumor growth of patient -derived xenografts via activation of p53 and inhibition of AKT.

The nucleophosmin 1 (NPM1) protein is frequently overexpressed in various cancers compared to normal tissues and represents a potential biomarker for maliganancy. However, its role in colorectal cancer (CRC) is still not fully understood. In this report, we show that NPM1 levels in CRC correlate with prognosis and sensitivity to chemotherapy. NPM1 expression was found to be significantly increased in CRC tumors (P < .001) and was associated with poor overall 5-year survival (P < .05). For individuals with Stage IV disease, this represented a reduction in survival by 11 months (P < .01; HR = 0.38, CI [0.21, 0.69]. In vitro, we show that NPM1 gene silencing enhanced the chemosensitivity of CRC cells and that pharmacological inhibition of NPM1 by NSC348884 triggered the onset of programmed cell death. Our immunofluorescence microscopy and immunoblot analyses also revealed that blocking NPM1 function sensitized CRC cells to chemotherapy-induced apoptosis through a mechanism that involves proteins in the AKT pathway. Consistent with the in vitro data, our patient-derived CRC xenograft model showed that inhibition of NPM1 suppressed tumor growth and attenuated AKT signaling in vivo. Moreover, LY294002, an inhibitor of the PI3K/AKT pathway, restored the chemosensitivity of CRC cells expressing high levels of NPM1. The findings that NPM1's expression in CRC tissue correlates with prognosis and supports anti-apoptotic activity mediated by AKT signaling, further our understanding of the role of NPM1 in CRC.

The interaction between SPARC and GRP78 interferes with ER stress signaling and potentiates apoptosis via PERK/eIF2α and IRE1α/XBP-1 in colorectal cancer.

Therapy-refractory disease is one of the main contributors of treatment failure in cancer. In colorectal cancer (CRC), SPARC can function as a sensitizer to conventional chemotherapy by enhancing apoptosis by interfering with the activity of Bcl-2. Here, we examine a novel mechanism by which SPARC further potentiates apoptosis via its modulation of the unfolded protein response (UPR). Using mass spectrometry to identify SPARC-associated proteins, GRP78 was identified as a protein partner for SPARC in CRC. In vitro studies conducted to assess the signaling events resulting from this interaction, included induction of ER stress with tunicamycin, 5-fluorouracil (5-FU), and irinotecan (CPT-11). We found that the interaction between GRP78 and SPARC increased during exposure to 5-FU, CPT-11, and tunicamycin, resulting in an attenuation of GRP78's inhibition of apoptosis. In addition, we also show that SPARC can sensitize CRC cells to PERK/eIF2α and IRE1α/XBP-1 UPR signaling by interfering with ER stress following binding to GRP78, which leads to ER stress-associated cell death in CRC cells. In line with these findings, a lower expression of GRP78 relative to SPARC in CRC is associated with a lower IC50 for 5-FU in either sensitive or therapy-refractory CRC cells. Interestingly, this observation correlates with tissue microarray analysis of 143 human CRC, where low GRP78 to SPARC expression level was prognostic of higher survival rate (P = 0.01) in individuals with CRC. This study demonstrates that modulation of UPR signaling by SPARC promotes ER stress-associated death and potentiates apoptosis. This may be an effective strategy that can be combined with current treatment options to improve therapeutic efficacy in CRC.

Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice.

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.

A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly.

The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.

EscE and EscG are cochaperones for the type III needle protein EscF of enteropathogenic Escherichia coli.

Type III secretion systems (T3SSs) are central virulence mechanisms used by a variety of Gram-negative bacteria to inject effector proteins into host cells. The needle polymer is an essential part of the T3SS that provides the effector proteins a continuous channel into the host cytoplasm. It has been shown for a few T3SSs that two chaperones stabilize the needle protein within the bacterial cytosol to prevent its premature polymerization. In this study, we characterized the chaperones of the enteropathogenic Escherichia coli (EPEC) needle protein EscF. We found that Orf2 and Orf29, two poorly characterized proteins encoded within the EPEC locus of enterocyte effacement (LEE), function as the needle protein cochaperones. Our finding demonstrated that both Orf2 and Orf29 are essential for type III secretion (T3S). In addition, we found that Orf2 and Orf29 associate with the bacterial membrane and form a complex with EscF. Orf2 and Orf29 were also shown to disrupt the polymerization of EscF in vitro. Prediction of the tertiary structures of Orf2 and Orf29 showed high structural homology to chaperones of other T3SS needle proteins. Overall, our data suggest that Orf2 and Orf29 function as the chaperones of the needle protein, and therefore, they have been renamed EscE and EscG.

Nucleophosmin 1, upregulated in adenomas and cancers of the colon, inhibits p53-mediated cellular senescence.

Dysregulation of nucleophosmin 1 (NPM1) has been found in numerous solid and hematological malignancies. Our previous meta-analysis of colorectal cancer (CRC) high throughput gene expression profiling studies identified it as a consistently reported up-regulated gene in the malignant state. Our aims were to compare NPM1 expression in normal colon, adenoma and CRC, to correlate their expressions with clinico-pathological parameters, and to assess the biological role of aberrant NPM1 expression in CRC cells. NPM1 transcript levels were studied in human CRC cell lines, whereas a tissue microarray of 57 normal human colon, 40 adenoma and 185 CRC samples were used to analyze NPM1 protein expression by immunohistochemistry. CRC cell lines were subjected to transient siRNA-mediated knockdown to study NPM1's roles on cell viability and senescence. NPM1 transcript levels were 7-11-folds higher in three different human CRC cell lines compared to normal colon cells. NPM1 protein expression was found to be progressively and significantly upregulated in CRC compared to adenomas and in adenomas compared to normal mucosa. Reducing NPM1 expression by siRNA had caused a significant decrease in cell viability, a concomitant increase in cellular senescence and cell cycle arrest. Cellular senescence induced under conditions of forced NPM1 suppression could be prevented by knocking down p53. The differential expression of NPM1 along the normal colon-adenoma-carcinoma progression and its involvement in resisting p53 related senescent growth arrest in CRC cell lines implicate its role in supporting CRC tumorigenesis.

Structural insight into the bacterial mucinase StcE essential to adhesion and immune evasion during enterohemorrhagic E. coli infection.

Mucin glycoproteins with large numbers of O-linked glycosylations comprise the mucosal barrier lining the mammalian gastrointestinal tract from mouth to gut. A critical biological function of mucins is to protect the underlying epithelium from infection. Enterohemorrhagic Escherichia coli (EHEC), the mediator of severe food- and water-borne disease, can breach this barrier and adhere to intestinal cells. StcE, a ∼100 kDa metalloprotease secreted by EHEC, plays a pivotal role in remodeling the mucosal lining during infection. To obtain mechanistic insight into its function, we have determined the structure of StcE. Our data reveal a dynamic, multidomain architecture featuring an unusually large substrate-binding cleft and a prominent polarized surface charge distribution highly suggestive of an electrostatic role in substrate targeting. The observation of key conserved motifs in the active site allows us to propose the structural basis for the specific recognition of α-O-glycan-containing substrates. Complementary biochemical analysis provides further insight into its distinct substrate specificity and binding stoichiometry.

A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system.

The type III secretion system (T3SS) is a macromolecular 'injectisome' that allows bacterial pathogens to transport virulence proteins into the eukaryotic host cell. This macromolecular complex is composed of connected ring-like structures that span both bacterial membranes. The crystal structures of the periplasmic domain of the outer membrane secretin EscC and the inner membrane protein PrgH reveal the conservation of a modular fold among the three proteins that form the outer membrane and inner membrane rings of the T3SS. This leads to the hypothesis that this conserved fold provides a common ring-building motif that allows for the assembly of the variably sized outer membrane and inner membrane rings characteristic of the T3SS. Using an integrated structural and experimental approach, we generated ring models for the periplasmic domain of EscC and placed them in the context of the assembled T3SS, providing evidence for direct interaction between the outer membrane and inner membrane ring components and an unprecedented span of the outer membrane secretin.