Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

AbstractThe regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca2+-dependent fashion. Molecular docking and conformational sampling studies of the Ca2+-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.Structured summary of protein interactionsCalbindin-D28K and Caspase-3 bind by isothermal titration calorimetry (View interaction

Long-term forest soil warming alters microbial communities in temperate forest soils

Soil microbes are major drivers of soil carbon cycling, yet we lack an understanding of how climate warming will affect microbial communities. Three ongoing field studies at the Harvard Forest Long-term Ecological Research (LTER) site (Petersham, MA) have warmed soils 5°C above ambient temperatures for 5, 8, and 20 years. We used this chronosequence to test the hypothesis that soil microbial communities have changed in response to chronic warming. Bacterial community composition was studied using Illumina sequencing of the 16S ribosomal RNA gene, and bacterial and fungal abundance were assessed using quantitative PCR. Only the 20-year warmed site exhibited significant change in bacterial community structure in the organic soil horizon, with no significant changes in the mineral soil. The dominant taxa, abundant at 0.1% or greater, represented 0.3% of the richness but nearly 50% of the observations (sequences). Individual members of the Actinobacteria, Alphaproteobacteria and Acidobacteria showed strong warming responses, with one Actinomycete decreasing from 4.5 to 1% relative abundance with warming. Ribosomal RNA copy number can obfuscate community profiles, but is also correlated with maximum growth rate or trophic strategy among bacteria. Ribosomal RNA copy number correction did not affect community profiles, but rRNA copy number was significantly decreased in warming plots compared to controls. Increased bacterial evenness, shifting beta diversity, decreased fungal abundance and increased abundance of bacteria with low rRNA operon copy number, including Alphaproteobacteria and Acidobacteria, together suggest that more or alternative niche space is being created over the course of long-term warming

Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa

Specificity of Molecular Fragments Binding to S100B versus S100A1 as Identified by NMR and Site Identification by Ligand Competitive Saturation (SILCS)

S100B, a biomarker of malignant melanoma, interacts with the p53 protein and diminishes its tumor suppressor function, which makes this S100 family member a promising therapeutic target for treating malignant melanoma. However, it is a challenge to design inhibitors that are specific for S100B in melanoma versus other S100-family members that are important for normal cellular activities. For example, S100A1 is most similar in sequence and structure to S100B, and this S100 protein is important for normal skeletal and cardiac muscle function. Therefore, a combination of NMR and computer aided drug design (CADD) was used to initiate the design of specific S100B inhibitors. Fragment-based screening by NMR, also termed “SAR by NMR,” is a well-established method, and was used to examine spectral perturbations in 2D [1H, 15N]-HSQC spectra of Ca2+-bound S100B and Ca2+-bound S100A1, side-by-side, and under identical conditions for comparison. Of the 1000 compounds screened, two were found to be specific for binding Ca2+-bound S100A1 and four were found to be specific for Ca2+-bound S100B, respectively. The NMR spectral perturbations observed in these six data sets were then used to model how each of these small molecule fragments showed specificity for one S100 versus the other using a CADD approach termed Site Identification by Ligand Competitive Saturation (SILCS). In summary, the combination of NMR and computational approaches provided insight into how S100A1 versus S100B bind small molecules specifically, which will enable improved drug design efforts to inhibit elevated S100B in melanoma. Such a fragment-based approach can be used generally to initiate the design of specific inhibitors for other highly homologous drug targets

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI

Characterizing inhibitors of human AP endonuclease 1

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme

Long-term forest soil warming alters microbial communities in temperate forest soils

Soil microbes are major drivers of soil carbon cycling, yet we lack an understanding of how climate warming will affect microbial communities. Three ongoing field studies at the Harvard Forest Long-term Ecological Research (LTER) site (Petersham, MA) have warmed soils 5°C above ambient temperatures for 5, 8, and 20 years. We used this chronosequence to test the hypothesis that soil microbial communities have changed in response to chronic warming. Bacterial community composition was studied using Illumina sequencing of the 16S ribosomal RNA gene, and bacterial and fungal abundance were assessed using quantitative PCR. Only the 20-year warmed site exhibited significant change in bacterial community structure in the organic soil horizon, with no significant changes in the mineral soil. The dominant taxa, abundant at 0.1% or greater, represented 0.3% of the richness but nearly 50% of the observations (sequences). Individual members of the Actinobacteria, Alphaproteobacteria and Acidobacteria showed strong warming responses, with one Actinomycete decreasing from 4.5 to 1% relative abundance with warming. Ribosomal RNA copy number can obfuscate community profiles, but is also correlated with maximum growth rate or trophic strategy among bacteria. Ribosomal RNA copy number correction did not affect community profiles, but rRNA copy number was significantly decreased in warming plots compared to controls. Increased bacterial evenness, shifting beta diversity, decreased fungal abundance and increased abundance of bacteria with low rRNA operon copy number, including Alphaproteobacteria and Acidobacteria, together suggest that more or alternative niche space is being created over the course of long-term warming

Long-term forest soil warming alters microbial communities in temperate forest soils

Soil microbes are major drivers of soil carbon cycling, yet we lack an understanding of how climate warming will affect microbial communities. Three ongoing field studies at the Harvard Forest Long-term Ecological Research (LTER) site (Petersham, MA) have warmed soils 5°C above ambient temperatures for 5, 8, and 20 years. We used this chronosequence to test the hypothesis that soil microbial communities have changed in response to chronic warming. Bacterial community composition was studied using Illumina sequencing of the 16S ribosomal RNA gene, and bacterial and fungal abundance were assessed using quantitative PCR. Only the 20-year warmed site exhibited significant change in bacterial community structure in the organic soil horizon, with no significant changes in the mineral soil. The dominant taxa, abundant at 0.1% or greater, represented 0.3% of the richness but nearly 50% of the observations (sequences). Individual members of the Actinobacteria, Alphaproteobacteria and Acidobacteria showed strong warming responses, with one Actinomycete decreasing from 4.5 to 1% relative abundance with warming. Ribosomal RNA copy number can obfuscate community profiles, but is also correlated with maximum growth rate or trophic strategy among bacteria. Ribosomal RNA copy number correction did not affect community profiles, but rRNA copy number was significantly decreased in warming plots compared to controls. Increased bacterial evenness, shifting beta diversity, decreased fungal abundance and increased abundance of bacteria with low rRNA operon copy number, including Alphaproteobacteria and Acidobacteria, together suggest that more or alternative niche space is being created over the course of long-term warming