Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin.

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally similar AB5-type protein toxins. They move from the cell surface to the endoplasmic reticulum where the A1 catalytic subunit is separated from its holotoxin by protein disulfide isomerase (PDI), thus allowing the dissociated A1 subunit to enter the cytosol for a toxic effect. Despite similar mechanisms of toxicity, CT is more potent than LT. The difference has been attributed to a more stable domain assembly for CT as compared to LT, but this explanation has not been directly tested and is arguable as toxin disassembly is an indispensable step in the cellular action of these toxins. We show here that PDI disassembles CT more efficiently than LT, which provides a possible explanation for the greater potency of the former toxin. Furthermore, direct examination of CT and LT domain assemblies found no difference in toxin stability. Using novel analytic geometry approaches, we provide a detailed characterization of the positioning of the A subunit with respect to the B pentamer and demonstrate significant differences in the interdomain architecture of CT and LT. Protein docking analysis further suggests that these global structural differences result in distinct modes of PDI-toxin interactions. Our results highlight previously overlooked structural differences between CT and LT that provide a new model for the PDI-assisted disassembly and differential potency of these toxins.

Model Combining Tumor Molecular and Clinicopathologic Risk Factors Predicts Sentinel Lymph Node Metastasis in Primary Cutaneous Melanoma.

PURPOSE: More than 80% of patients who undergo sentinel lymph node (SLN) biopsy have no nodal metastasis. Here we describe a model that combines clinicopathologic and molecular variables to identify patients with thin and intermediate thickness melanomas who may forgo the SLN biopsy procedure due to their low risk of nodal metastasis. PATIENTS AND METHODS: Genes with functional roles in melanoma metastasis were discovered by analysis of next generation sequencing data and case control studies. We then used PCR to quantify gene expression in diagnostic biopsy tissue across a prospectively designed archival cohort of 754 consecutive thin and intermediate thickness primary cutaneous melanomas. Outcome of interest was SLN biopsy metastasis within 90 days of melanoma diagnosis. A penalized maximum likelihood estimation algorithm was used to train logistic regression models in a repeated cross validation scheme to predict the presence of SLN metastasis from molecular, clinical and histologic variables. RESULTS: Expression of genes with roles in epithelial-to-mesenchymal transition (glia derived nexin, growth differentiation factor 15, integrin ß3, interleukin 8, lysyl oxidase homolog 4, TGFß receptor type 1 and tissue-type plasminogen activator) and melanosome function (melanoma antigen recognized by T cells 1) were associated with SLN metastasis. The predictive ability of a model that only considered clinicopathologic or gene expression variables was outperformed by a model which included molecular variables in combination with the clinicopathologic predictors Breslow thickness and patient age; AUC, 0.82; 95% CI, 0.78-0.86; SLN biopsy reduction rate of 42% at a negative predictive value of 96%. CONCLUSION: A combined model including clinicopathologic and gene expression variables improved the identification of melanoma patients who may forgo the SLN biopsy procedure due to their low risk of nodal metastasis.

Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site.

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also L-fucose alone, bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called 'secretors'.

FAK auto-phosphorylation site tyrosine 397 is required for development but dispensable for normal skin homeostasis.

Focal adhesion kinase (FAK) is an intensely studied non-receptor tyrosine kinase with roles in cancer and other common human diseases. Despite the large interest in FAK, the in vivo contribution of FAK auto-phosphorylation site tyrosine (Y) 397 to FAK function is incompletely understood. To study FAK Y397 in vivo we analyzed mice with 'non-phosphorylatable' Y-to-phenylalanine (F) and 'phospho-mimicking' Y-to-glutamate (E) mutations in the germline. We found that FAK Y397F mice die early during embryogenesis with abnormal angiogenesis like FAK kinase-dead mice. When Y397 is mutated to a glutamate mice survive beyond mid-gestation like mice where Y397 is lost by deletion of FAK exon 15. In culture, defects in proliferation, invasion and gene expression were more severe with the FAK Y397F than with the FAK Y397E mutation despite the inability of FAK Y397E to bind SRC. Conditional expression of FAK Y397F or Y397E in unchallenged avascular epidermis, however, resulted in no appreciable phenotype. We conclude that FAK Y397 is required for the highly dynamic tissue remodeling during development but dispensable for normal homeostasis of avascular epidermis. In contrast to the Y397F mutation, FAK Y397E retains sufficient biological activity to allow for development beyond mid-gestation.

Inter-Enzyme Allosteric Regulation of Chorismate Mutase in Corynebacterium glutamicum: Structural Basis of Feedback Activation by Trp.

Corynebacterium glutamicum is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM-CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from Mycobacterium tuberculosis.

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)-kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.

Tumor Cell Adhesion As a Risk Factor for Sentinel Lymph Node Metastasis in Primary Cutaneous Melanoma.

PURPOSE: Less than 20% of patients with melanoma who undergo sentinel lymph node (SLN) biopsy based on American Society of Clinical Oncology/Society of Surgical Oncology recommendations are SLN positive. We present a multi-institutional study to discover new molecular risk factors associated with SLN positivity in thin and intermediate-thickness melanoma. PATIENTS AND METHODS: Gene clusters with functional roles in melanoma metastasis were discovered by next-generation sequencing and validated by quantitative polymerase chain reaction using a discovery set of 73 benign nevi, 76 primary cutaneous melanoma, and 11 in-transit melanoma metastases. We then used polymerase chain reaction to quantify gene expression in a model development cohort of 360 consecutive thin and intermediate-thickness melanomas and a validation cohort of 146 melanomas. Outcome of interest was SLN biopsy metastasis within 90 days of melanoma diagnosis. Logic and logistic regression analyses were used to develop a model for the likelihood of SLN metastasis from molecular, clinical, and histologic variables. RESULTS: ITGB3, LAMB1, PLAT, and TP53 expression were associated with SLN metastasis. The predictive ability of a model that included these molecular variables in combination with clinicopathologic variables (patient age, Breslow depth, and tumor ulceration) was significantly greater than a model that only considered clinicopathologic variables and also performed well in the validation cohort (area under the curve, 0.93; 95% CI, 0.87 to 0.97; false-positive and false-negative rates of 22% and 0%, respectively, using a 10% cutoff for predicted SLN metastasis risk). CONCLUSION: The addition of cell adhesion-linked gene expression variables to clinicopathologic variables improves the identification of patients with SLN metastases within 90 days of melanoma diagnosis.

Vinylogous chain branching catalysed by a dedicated polyketide synthase module.

Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like ß-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching-acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.

Structure-based engineering increased the catalytic turnover rate of a novel phenazine prenyltransferase.

Prenyltransferases (PTs) catalyze the regioselective transfer of prenyl moieties onto aromatic substrates in biosynthetic pathways of microbial secondary metabolites. Therefore, these enzymes contribute to the chemical diversity of natural products. Prenylation is frequently essential for the pharmacological properties of these metabolites, including their antibiotic and antitumor activities. Recently, the first phenazine PTs, termed EpzP and PpzP, were isolated and biochemically characterized. The two enzymes play a central role in the biosynthesis of endophenazines by catalyzing the regiospecific prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dhPCA) in the secondary metabolism of two different Streptomyces strains. Here we report crystal structures of EpzP in its unliganded state as well as bound to S-thiolodiphosphate (SPP), thus defining the first three-dimensional structures for any phenazine PT. A model of a ternary complex resulted from in silico modeling of dhPCA and site-directed mutagenesis. The structural analysis provides detailed insight into the likely mechanism of phenazine prenylation. The catalytic mechanism suggested by the structure identifies amino acids that are required for catalysis. Inspection of the structures and the model of the ternary complex furthermore allowed us to rationally engineer EpzP variants with up to 14-fold higher catalytic reaction rate compared to the wild-type enzyme. This study therefore provides a solid foundation for additional enzyme modifications that should result in efficient, tailor-made biocatalysts for phenazines production.