Explore the vast range of titles available.

The structure of C3b in complex with factor I and a shortened version of factor H, along with functional analyses, leads to a mechanistic model for how regulators determine sequential cleavage events on C3b. The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1–4 with 19–20). FI binds C3b–FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

The evolution of chemical signals is subject to environmental constraints. A multicomponent signal may combine semiochemical molecules with supporting compounds able to enhance communication efficacy. Carbonic anhydrases (CAs) are ubiquitous enzymes catalysing the reversible hydration of carbon dioxide, a reaction involved in a variety of physiological processes as it controls the chemical environment of the different tissues or cellular compartments, thus contributing to the overall system homeostasis. CA-IV isoform has been recently identified by mass spectrometry in the femoral gland secretions (FG) of the marine iguana, where it has been hypothesized to contribute to the chemical stability of the signal, by regulating blend pH. Lizards, indeed, use FG to communicate by delivering the waxy secretion on bare substrate, where it is exposed to environmental stressors. Therefore, we expect that some molecules in the mixture may play supporting functions, enhancing the stability of the chemical environment, or even conferring homeostatic properties to the blend. CA-IV may well represent an important candidate to this hypothesized supporting/homeostatic function, and, therefore, we can expect it to be common in FG secretions of other lizard species. To evaluate this prediction and definitely validate CA identity, we analysed FG secretions of eight species of wall lizards (genus Podarcis ), combining mass spectrometry, immunoblotting, immunocytochemistry, and transmission electron microscopy. We demonstrate CA-IV to actually occur in the FG of seven out of the eight considered species, providing an immunochemistry validation of mass-spectrometry identifications, and localizing the enzyme within the secretion mass. The predicted structure of the identified CA is compatible with the known enzymatic activity of CA-IV, supporting the hypothesis that CA play a signal homeostasis function and opening to new perspective about the role of proteins in vertebrate chemical communication.

Lysyl hydroxylases catalyze hydroxylation of collagen lysines, and sustain essential roles in extracellular matrix (ECM) maturation and remodeling. Malfunctions in these enzymes cause severe connective tissue disorders. Human lysyl hydroxylase 3 (LH3/PLOD3) bears multiple enzymatic activities, as it catalyzes collagen lysine hydroxylation and also their subsequent glycosylation. Our understanding of LH3 functions is currently hampered by lack of molecular structure information. Here, we present high resolution crystal structures of full-length human LH3 in complex with cofactors and donor substrates. The elongated homodimeric LH3 architecture shows two distinct catalytic sites at the N- and C-terminal boundaries of each monomer, separated by an accessory domain. The glycosyltransferase domain displays distinguishing features compared to other known glycosyltransferases. Known disease-related mutations map in close proximity to the catalytic sites. Collectively, our results provide a structural framework characterizing the multiple functions of LH3, and the molecular mechanisms of collagen-related diseases involving human lysyl hydroxylases. Lysyl hydroxylase 3 (LH3) catalyzes collagen lysine hydroxylation and their subsequent O-linked glycosylation. Here the authors provide mechanistic insights into the lysyl hydroxylase and glycosyltransferase activities of LH3 by determining the crystal structures of full-length human LH3 bound to cofactors and donor substrates.

During collagen biosynthesis, lysine residues undergo extensive post-translational modifications through the alternate action of two distinct metal ion-dependent enzyme families (i.e., LH/PLODs and GLT25D/COLGALT), ultimately producing the highly conserved α-(1,2)-glucosyl-β-(1,O)-galactosyl-5-hydroxylysine pattern. Malfunctions in these enzymes are linked to developmental pathologies and extracellular matrix alterations associated to enhanced aggressiveness of solid tumors. Here, we characterized human GLT25D1/COLGALT1, revealing an elongated head-to-head homodimeric assembly. Each monomer encompasses two domains (named GT1 and GT2), both unexpectedly capable of binding metal ion cofactors and UDP-α-galactose donor substrates, resulting in four candidate catalytic sites per dimer. We identify the catalytic site in GT2, featuring an unusual Glu-Asp-Asp motif critical for Mn 2+ binding, ruling out direct catalytic roles for the GT1 domain, but showing that in this domain the unexpectedly bound Ca 2+ and UDP-α-galactose cofactors are critical for folding stability. Dimerization, albeit not essential for GLT25D1/COLGALT1 activity, provides a critical molecular contact site for multi-enzyme assembly interactions with partner multifunctional LH/PLOD lysyl hydroxylase-glycosyltransferase enzymes. Hydroxylysine galactosylation is essential for collagen maturation. This work reveals the molecular structure of human GLT25D1, its catalytic residues, and a noncatalytic structural domain stabilized by metal ion and donor substrate binding.

With its noncatalytic domains, DNA-binding regions, and a catalytic core targeting the histone tails, LSD1-CoREST (lysine-specific demethylase 1; REST corepressor) is an ideal model system to study the interplay between DNA binding and histone modification in nucleosome recognition. To this end, we covalently associated LSD1-CoREST to semisynthetic nucleosomal particles. This enabled biochemical and biophysical characterizations of nucleosome binding and structural elucidation by small-angle X-ray scattering, which was extensively validated through binding assays and site-directed mutagenesis of functional interfaces. Our results suggest that LSD1-CoREST functions as an ergonomic clamp that induces the detachment of the H3 histone tail from the nucleosomal DNA to make it available for capture by the enzyme active site. The key notion emerging from these studies is the inherently competitive nature of the binding interactions because nucleosome tails, chromatin modifiers, transcription factors, and DNA represent sites for multiple and often mutually exclusive interactions. Significance The correct and regulated readout of epigenetic marks on chromatin is essential to modulate gene expression in living cells. The regulation of chromatin accessibility is ensured by such epigenetic tags, which form a platform for the binding of specific enzymatic modules. A clear example of this mechanism is represented by the histone demethylase LSD1-CoREST, which removes methylation marks from lysine 4 of histone protein H3. We developed a crosslinking technology to capture this histone demethylase in contact with the nucleosome and used this methodology to explore the structural and biophysical properties of this complex. This is one of the very few successful attempts to visualize the molecular mechanism underlying the recognition of the nucleosomal substrate by a histone-modifying enzyme complex.

In autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need. Here, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A receptor (PLA R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker. BiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig and T cells. , the BiAATE successfully induced T cell-dependent depletion of PLA R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA R antibody levels following active immunization with PLA R. Should this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B’s necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis. UNC5B is a Netrin-1 receptor expressed in endothelial cells that in the absence of ligand induces apoptosis. Here the authors identify an UNC5B splicing isoform that is insensitive to the pro-survival ligand Netrin-1 and is required for apoptosis-dependent blood vessel development.

Many cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound. Here, we explore the different binding strategies and chemical tricks that enzymes have developed to overcome this problem. These reactions can be catalyzed by integral membrane proteins that channel a hydrophilic molecule into their active site, as well as by water-soluble enzymes that are able to capture a lipophilic substrate from the phospholipid bilayer. Many chemical and biological aspects of this type of enzymology remain to be investigated and will require the integration of protein chemistry with membrane biology.

Background Mosquito-borne arboviral diseases represent a growing threat and serious worldwide concern for public health authorities. Host immunoglobulin G (IgG) responses to mosquito salivary antigens emerged as a useful additional tool to evaluate human–vector contact, which is crucial for transmission risk assessment and planning vector control interventions. We previously reported that IgG responses to the Aedes albopictus 34k2 salivary protein (al34k2) are suitable, although with some limitations, to reveal variation of human exposure to the tiger mosquito. In this study we evaluated the Ae. albopictus Ag5-3 (alAg5-3), an Antigen 5 family member specifically and abundantly expressed in the saliva of adult females. Methods IgG responses to recombinant alAg5-3, as well as to a combination of alAg5-3 and al34k2, were measured in a set of sera previously collected from healthy human blood donors before and after the summer season of exposure to mosquito bites. Surveys were conducted in two districts of Northeast Italy, Padua and Belluno, with different density and history of colonization by the tiger mosquito Ae. albopictus . Results A preliminary pilot study, performed on a small subset of individuals from Padua, indicated that alAg5-3 was more immunogenic than al34k2 and may be suitable to detect variations of exposure to Ae. albopictus . Analysis of the whole set of 523 sera showed that anti-alAg5-3 IgG levels significantly increased, in both study areas, after the summer period of high mosquito density. However, differences between the two study sites were only found when a mixture of the two antigens, alAg5-3 and al34k2, was used. Conclusions IgG responses to alAg5-3 represent a novel appropriate marker to evaluate seasonal variation of human exposure to Ae. albopictus and, because of its higher sensitivity, it appears preferable to al34k2, especially for longitudinal studies in conditions of low-to-moderate mosquito density. However, the combination of both antigens may be a better surrogate of Ae. albopictus saliva since it allows the detection of both temporal and spatial variations of exposure to Ae. albopictus bites. The high conservation of the Ag5-3 protein among Aedes species suggests it may be exploited to also reveal exposure to Aedes aegypti and perhaps to other Aedes species. Graphical Abstract

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies. Growing tumors stimulate the formation of new blood vessels to supply the oxygen and nutrients the cancerous cells need to stay alive. Stopping tumors from forming the blood vessels could therefore help us to treat cancer. To do so, we need to understand how different proteins control when and how blood vessels develop. Cells make proteins by first ‘transcribing’ genes to form RNA molecules. In many cases, the RNA then goes through a process called alternative splicing. Proteins known as splicing factors cut out different segments of the RNA molecule and stick together the remaining segments to form templates for protein production. This enables a single gene to produce many different variants of a protein. Angiolini, Belloni, Giordano et al. have now studied mouse and human versions of the cells that line the blood vessels grown by tumors. This revealed that a splicing factor called NOVA2 targets a protein called L1CAM, which is normally responsible for gluing adjacent cells together. Angiolini et al. found that NOVA2 splices L1CAM into a form not seen before. Instead of remaining anchored to cell surfaces, the newly identified form of L1CAM is released into the blood circulation, where it stimulates new blood vessels to grow. Samples taken from the blood vessels of human ovarian tumors showed high levels of both NOVA2 and the modified form of L1CAM, while blood vessels in healthy tissue contain no, or very low levels of both proteins. Therefore, if the new form of L1CAM can be detected in the blood, it could be used to help cancer diagnosis, and to indicate which patients would benefit from treatments that restrict the growth of blood vessels in tumors. Further work is now needed to explore these possibilities.