Hereditary cancer-associated missense mutations in hMSH6 uncouple ATP hydrolysis from DNA mismatch binding

Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.     

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.     

Manipulation of fungal development as source of novel secondary metabolites for biotechnology

Fungal genomics revealed a large potential of yet-unexplored secondary metabolites, which are not produced during vegetative growth. The discovery of novel bioactive compounds is increasingly gaining importance. The high number of resistances against established antibiotics requires novel drugs to counteract increasing human and animal mortality rates. In addition, growth of plant pathogens has to be controlled to minimize harvest losses. An additional critical issue is the post-harvest production of deleterious mycotoxins. Fungal development and secondary metabolite production are linked processes. Therefore, molecular regulators of development might be suitable to discover new bioactive fungal molecules or to serve as targets to control fungal growth, development, or secondary metabolite production. The fungal impact is relevant as well for our healthcare systems as for agriculture. We propose here to use the knowledge about mutant strains discovered in fungal model systems for a broader application to detect and explore new fungal drugs or toxins. As examples, mutant strains impaired in two conserved eukaryotic regulatory complexes are discussed. The COP9 signalosome (CSN) and the velvet complex act at the interface between development and secondary metabolism. The CSN is a multi-protein complex of up to eight subunits and controls the activation of CULLIN-RING E3 ubiquitin ligases, which mark substrates with ubiquitin chains for protein degradation by the proteasome. The nuclear velvet complex consists of the velvet-domain proteins VeA and VelB and the putative methyltransferase LaeA acting as a global regulator for secondary metabolism. Defects in both complexes disturb fungal development, light perception, and the control of secondary metabolism. The potential biotechnological relevance of these developmental fungal mutant strains for drug discovery, agriculture, food safety, and human healthcare is discussed.     

Incidence,Trends and Ethnic Differences of Oropharyngeal,Anal and Cervical Cancers: Singapore, 1968-2012

In recent decades, several Western countries have reported an increase in oropharyngeal and anal cancers caused by human papillomavirus (HPV). Trends in HPV-associated cancers in Asia have not been as well described. We describe the epidemiology of potentially HPV-related cancers reported to the Singapore Cancer Registry from 1968–2012. Analysis included 998 oropharyngeal squamous cell carcinoma (OPSCC), 183 anal squamous cell carcinoma (ASCC) and 8,019 invasive cervical cancer (ICC) cases. Additionally, 368 anal non-squamous cell carcinoma (ANSCC) and 2,018 non-oropharyngeal head and neck carcinoma (non-OP HNC) cases were included as comparators. Age-standardized incidence rates (ASR) were determined by gender and ethnicity (Chinese, Malay and Indian). Joinpoint regression was used to evaluate annual percentage change (APC) in incidence. OPSCC incidence increased in both genders (men 1993–2012, APC = 1.9%, p<0.001; women 1968–2012, APC = 2.0%, p = 0.01) and was 5 times higher in men than women. In contrast, non-OP HNC incidence declined between 1968–2012 among men (APC = -1.6%, p<0.001) and women (APC = -0.4%, p = 0.06). ASCC and ANSCC were rare (ASR = 0.2 and 0.7 per 100,000 person-years, respectively) and did not change significantly over time except for increasing ANSCCs in men (APC = 2.8%, p<0.001). ICC was the most common HPV-associated cancer (ASR = 19.9 per 100,000 person-years) but declined significantly between 1968–2012 (APC = -2.4%). Incidence of each cancer varied across ethnicities. Similar to trends in Western countries, OPSCC incidence increased in recent years, while non-OP HNC decreased. ICC remains the most common HPV-related cancer in Singapore, but Pap screening programs have led to consistently decreasing incidence.     

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.     

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.     

Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1). During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature. A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.