Developing methodologies for exploring myosin-5 elasticity under strain

Motor proteins drive the movement of organelles and other types of cargo inside every eukaryotic cell type. These motors include myosins, kinesins and dyneins. Myosin-5a, a ubiquitous motor protein, has been intensively studied and its enzymatic properties and cellular functions have been described in great detail. This dimeric molecule walks processively along actin filaments, enabling it to transport a variety of cargos including mRNA, pigment granules, organelles such as the endoplasmic reticulum and endocytic vesicles. Cargos bind to the C-terminal globular tail domain of the molecule, while the N-terminal motor domain hydrolyses ATP to take regular steps on the actin filament. The structure and enzymatic properties of myosin-5 moving processively along actin under unloaded conditions have already been well characterised. However, while pulling a cargo through the viscoelastic cytoplasm, the molecule will experience variable forces. Therefore it is important to study how these forces alter the structure and kinetic behaviour of myosin-5. The purpose of this study was to design myosin-5 constructs that could be tethered, in a controlled manner, to actin via sequences introduced into the tail region of the molecule. The tethered motor protein is expected to ’stall’ while moving along the actin filament. In this way, the effects of strain on the myosin-5 structure and kinetics could then be investigated. This would simulate a case where inside the cell myosin-5 stalls due to strain, for example, whilst trying to move its cargo through spatially restricted areas of the actin cytoskeleton. To find a new way to attach the tail of myosin-5 to actin, small, artificial actin-binding proteins named Adhirons were raised with phage display assay and their properties were explored. They bind to actin with high affinity and they are also useful for staining actin in cells or for attaching actin to coverslips in various motility assays. Multiple myosin-5 constructs were explored, that had either an Avi-tag or an actin-binding Adhiron at their C-terminal ends. A construct that contained the full predicted coiled-coil motif of myosin-5, could not be attached to the same actin filament via its tail as the motors were bound to. Two further constructs that have an artificial long coiled-coil tail, were cloned and expression trials are currently in progress

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent

Keeping Allergen Names Clear and Defined

The World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee was established in 1986 by leading allergists to standardize names given to proteins that cause IgE-mediated reactions in humans. The Sub-Committee’s objective is to assign unique names to allergens based on a critical analysis of confidentially submitted biochemical and clinical data from researchers, often prior to publication to preserve consistency. The Sub-Committee maintains and revises the database as the understanding of allergens evolves. This report summarizes recent developments that led to updates in classification of cockroach group 1 and 5 allergens to animal as well as environmental and occupational allergens. Interestingly, routes, doses, and frequency of exposure often affects allergenicity as does the biochemical properties of the proteins and similarity to self and other proteins. Information required by the Sub-Committee now is more extensive than previously as technology has improved. Identification of new allergens requires identification of the amino acid sequence and physical characteristics of the protein as well as demonstration of IgE binding from subjects verified by described clinical histories, proof of the presence of the protein in relevant exposure substances, and demonstration of biological activity (skin prick tests, activation of basophils, or mast cells). Names are assigned based on taxonomy with the abbreviation of genus and species and assignment of a number, which reflects the priority of discovery, but more often now, the relationships with homologous proteins in related species

The dUTPase Enzyme Is Essential in Mycobacterium smegmatis

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. All mycobacterial genomes encode dUTPase with a mycobacteria-specific surface loop absent in the human dUTPase. Using Mycobacterium smegmatis as a fast growing model for Mycobacterium tuberculosis, we demonstrate that dUTPase knock-out results in lethality that can be reverted by complementation with wild-type dUTPase. Interestingly, a mutant dUTPase gene lacking the genus-specific loop was unable to complement the knock-out phenotype. We also show that deletion of the mycobacteria-specific loop has no major effect on dUTPase enzymatic properties in vitro and thus a yet to be identified loop-specific function seems to be essential within the bacterial cell context. In addition, here we demonstrated that Mycobacterium tuberculosis dUTPase is fully functional in Mycobacterium smegmatis as it rescues the lethal knock-out phenotype. Our results indicate the potential of dUTPase as a target for antitubercular drugs and identify a genus-specific surface loop on the enzyme as a selective target

Autistic behavior in boys with fragile X syndrome: social approach and HPA-axis dysfunction

The primary goal of this study was to examine environmental and neuroendocrine factors that convey increased risk for elevated autistic behavior in boys with Fragile X syndrome (FXS). This study involves three related analyses: (1) examination of multiple dimensions of social approach behaviors and how they vary over time, (2) investigation of mean levels and modulation of salivary cortisol levels in response to social interaction, and (3) examination of the relationship of social approach and autistic behaviors to salivary cortisol. Poor social approach and elevated baseline and regulation cortisol are discernible traits that distinguish boys with FXS and ASD from boys with FXS only and from typically developing boys. In addition, blunted cortisol change is associated with increased severity of autistic behaviors only within the FXS and ASD group. Boys with FXS and ASD have distinct behavioral and neuroendocrine profiles that differentiate them from those with FXS alone and typically developing boys

Ubiquitination in the ERAD process

In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation

Keeping Allergen Names Clear and Defined

The World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee was established in 1986 by leading allergists to standardize names given to proteins that cause IgE-mediated reactions in humans. The Sub-Committee’s objective is to assign unique names to allergens based on a critical analysis of confidentially submitted biochemical and clinical data from researchers, often prior to publication to preserve consistency. The Sub-Committee maintains and revises the database as the understanding of allergens evolves. This report summarizes recent developments that led to updates in classification of cockroach group 1 and 5 allergens to animal as well as environmental and occupational allergens. Interestingly, routes, doses, and frequency of exposure often affects allergenicity as does the biochemical properties of the proteins and similarity to self and other proteins. Information required by the Sub-Committee now is more extensive than previously as technology has improved. Identification of new allergens requires identification of the amino acid sequence and physical characteristics of the protein as well as demonstration of IgE binding from subjects verified by described clinical histories, proof of the presence of the protein in relevant exposure substances, and demonstration of biological activity (skin prick tests, activation of basophils, or mast cells). Names are assigned based on taxonomy with the abbreviation of genus and species and assignment of a number, which reflects the priority of discovery, but more often now, the relationships with homologous proteins in related species

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases.

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg(2+)-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80 A resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg(2+) accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function