Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2.

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Assessing the safety of new germicidal far-UVC technologies.

The COVID-19 pandemic underscored the crucial importance of enhanced indoor air quality control measures to mitigate the spread of respiratory pathogens. Far-UVC is a type of germicidal ultraviolet technology, with wavelengths between 200 and 235 nm, that has emerged as a highly promising approach for indoor air disinfection. Due to its enhanced safety compared to conventional 254 nm upper-room germicidal systems, far-UVC allows for whole-room direct exposure of occupied spaces, potentially offering greater efficacy, since the total room air is constantly treated. While current evidence supports using far-UVC systems within existing guidelines, understanding the upper safety limit is critical to maximizing its effectiveness, particularly for the acute phase of a pandemic or epidemic when greater protection may be needed. This review article summarizes the substantial present knowledge on far-UVC safety regarding skin and eye exposure and highlights research priorities to discern the maximum exposure levels that avoid adverse effects. We advocate for comprehensive safety studies that explore potential mechanisms of harm, generate action spectra for crucial biological effects and conduct high-dose, long-term exposure trials. Such rigorous scientific investigation will be key to determining safe and effective levels for far-UVC deployment in indoor environments, contributing significantly to future pandemic preparedness and response.

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM.

Development of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic strategy for Alzheimer's disease.

Recent genome-wide association studies have revealed genetic risk factors for Alzheimer's disease (AD) that are exclusively expressed in microglia within the brain. A proteomics approach identified moesin (MSN), a FERM (four-point-one ezrin radixin moesin) domain protein, and the receptor CD44 as hub proteins found within a co-expression module strongly linked to AD clinical and pathological traits as well as microglia. The FERM domain of MSN interacts with the phospholipid PIP2 and the cytoplasmic tails of receptors such as CD44. This study explored the feasibility of developing protein-protein interaction inhibitors that target the MSN-CD44 interaction. Structural and mutational analyses revealed that the FERM domain of MSN binds to CD44 by incorporating a beta strand within the F3 lobe. Phage-display studies identified an allosteric site located close to the PIP2 binding site in the FERM domain that affects CD44 binding within the F3 lobe. These findings support a model in which PIP2 binding to the FERM domain stimulates receptor tail binding through an allosteric mechanism that causes the F3 lobe to adopt an open conformation permissive for binding. High-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction, and one compound series was further optimized for biochemical activity, specificity, and solubility. The results suggest that the FERM domain holds potential as a drug development target. The small molecule preliminary leads generated from the study could serve as a foundation for additional medicinal chemistry effort with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Analysis of the first genetic engineering attribution challenge.

The ability to identify the designer of engineered biological sequences-termed genetic engineering attribution (GEA)-would help ensure due credit for biotechnological innovation, while holding designers accountable to the communities they affect. Here, we present the results of the first Genetic Engineering Attribution Challenge, a public data-science competition to advance GEA techniques. Top-scoring teams dramatically outperformed previous models at identifying the true lab-of-origin of engineered plasmid sequences, including an increase in top-1 and top-10 accuracy of 10 percentage points. A simple ensemble of prizewinning models further increased performance. New metrics, designed to assess a model's ability to confidently exclude candidate labs, also showed major improvements, especially for the ensemble. Most winning teams adopted CNN-based machine-learning approaches; however, one team achieved very high accuracy with an extremely fast neural-network-free approach. Future work, including future competitions, should further explore a wide diversity of approaches for bringing GEA technology into practical use.

Bidirectional contact tracing could dramatically improve COVID-19 control.

Contact tracing is critical to controlling COVID-19, but most protocols only "forward-trace" to notify people who were recently exposed. Using a stochastic branching-process model, we find that "bidirectional" tracing to identify infector individuals and their other infectees robustly improves outbreak control. In our model, bidirectional tracing more than doubles the reduction in effective reproduction number (Reff) achieved by forward-tracing alone, while dramatically increasing resilience to low case ascertainment and test sensitivity. The greatest gains are realised by expanding the manual tracing window from 2 to 6 days pre-symptom-onset or, alternatively, by implementing high-uptake smartphone-based exposure notification; however, to achieve the performance of the former approach, the latter requires nearly all smartphones to detect exposure events. With or without exposure notification, our results suggest that implementing bidirectional tracing could dramatically improve COVID-19 control.

Extreme genomic volatility characterizes the evolution of the immunoglobulin heavy chain locus in cyprinodontiform fishes.

The evolution of the adaptive immune system has provided vertebrates with a uniquely sophisticated immune toolkit, enabling them to mount precise immune responses against a staggeringly diverse range of antigens. Like other vertebrates, teleost fishes possess a complex and functional adaptive immune system; however, our knowledge of the complex antigen-receptor genes underlying its functionality has been restricted to a small number of experimental and agricultural species, preventing systematic investigation into how these crucial gene loci evolve. Here, we analyse the genomic structure of the immunoglobulin heavy chain (IGH) gene loci in the cyprinodontiforms, a diverse and important group of teleosts present in many different habitats across the world. We reconstruct the complete IGH loci of the turquoise killifish (Nothobranchius furzeri) and the southern platyfish (Xiphophorus maculatus) and analyse their in vivo gene expression, revealing the presence of species-specific splice isoforms of transmembrane IGHM. We further characterize the IGH constant regions of 10 additional cyprinodontiform species, including guppy, Amazon molly, mummichog and mangrove killifish. Phylogenetic analysis of these constant regions suggests multiple independent rounds of duplication and deletion of the teleost-specific antibody class IGHZ in the cyprinodontiform lineage, demonstrating the extreme volatility of IGH evolution. Focusing on the cyprinodontiforms as a model taxon for comparative evolutionary immunology, this work provides novel genomic resources for studying adaptive immunity and sheds light on the evolutionary history of the adaptive immune system.

Molecular features of lipoprotein CD0873: A potential vaccine against the human pathogen Clostridioides difficile.

Clostridioides difficile is the primary cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease resulting in a significant fatality rate. Colonization of the gut is critical for C. difficile pathogenesis. The bacterial molecules essential for efficient colonization therefore offer great potential as vaccine candidates. Here we present findings demonstrating that the C. difficile immunogenic lipoprotein CD0873 plays a critical role in pathogen success in vivo We found that in a dixenic colonization model, a CD0873-positive strain of C. difficile significantly outcompeted a CD0873-negative strain. Immunization of mice with recombinant CD0873 prevented long-term gut colonization and was correlated with a strong secretory IgA immune response. We further present high-resolution crystal structures of CD0873, at 1.35-2.50 Å resolutions, offering a first view of the ligand-binding pocket of CD0873 and provide evidence that this lipoprotein adhesin is part of a tyrosine import system, an amino acid key in C. difficile infection. These findings suggest that CD0873 could serve as an effective component in a vaccine against C. difficile.

The structure of the S-layer of Clostridium difficile.

The nosocomially acquired pathogen Clostridium difficile is the primary causative agent of antibiotic associated diarrhoea and causes tens of thousands of deaths globally each year. C. difficile presents a paracrystalline protein array on the surface of the cell known as an S-layer. S-layers have been demonstrated to possess a wide range of important functions, which, combined with their inherent accessibility, makes them a promising drug target. The unusually complex S-layer of C. difficile is primarily comprised of the high- and low- molecular weight S-layer proteins, HMW SLP and LMW SLP, formed from the cleavage of the S-layer precursor protein, SlpA, but may also contain up to 28 SlpA paralogues. A model of how the S-layer functions as a whole is required if it is to be exploited in fighting the bacterium. Here, we provide a summary of what is known about the S-layer of C. difficile and each of the paralogues and, considering some of the domains present, suggest potential roles for them.

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile.

Clostridium difficile is a burden to healthcare systems around the world, causing tens of thousands of deaths annually. The S-layer of the bacterium, a layer of protein found of the surface of cells, has received a significant amount of attention over the past two decades as a potential target to combat the growing threat presented by C. difficile infections. The S-layer contains a wide range of proteins, each of which possesses three cell wall-binding domains, while many also possess a "functional" region. Here, we present the high resolution structure of the functional region of one such protein, Cwp19 along with preliminary functional characterisation of the predicted glycoside hydrolase. Cwp19 has a TIM barrel fold and appears to possess a high degree of substrate selectivity. The protein also exhibits peptidoglycan hydrolase activity, an order of magnitude slower than that of lysozyme and is the first member of glycoside hydrolase-like family 10 to be characterised. This research goes some way to understanding the role of Cwp19 in the S-layer of C. difficile. DATABASE: Structural data are available in the PDB under the accession numbers 5OQ2 and 5OQ3.

Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro.

Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S-layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S-layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the 'functional' region of Cwp2, consisting of residues 29-318 at 1.9 Å, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2. DATABASES: Structural data are available in the PDB under the accession number 5NJL.

Structural insights into human angiogenin variants implicated in Parkinson's disease and Amyotrophic Lateral Sclerosis.

Mutations in Angiogenin (ANG), a member of the Ribonuclease A superfamily (also known as RNase 5) are known to be associated with Amyotrophic Lateral Sclerosis (ALS, motor neurone disease) (sporadic and familial) and Parkinson's Disease (PD). In our previous studies we have shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. In addition, in an extensive study on selective ANG-ALS variants we correlated the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. Furthermore, we have established that ANG-ALS variants which affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Here, we report a detailed experimental structural study on eleven new ANG-PD/ALS variants which will have implications in understanding the molecular basis underlying their role in PD and ALS.

Molecular features of the sortase enzyme family.

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram-positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram-positive bacteria, it is likely that other Gram-positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools.

Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide.

In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host-pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.

The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile.

Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Šresolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33-497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.