Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc.

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.

POC1A, prognostic biomarker of immunosuppressive microenvironment in cancer.

POC1 centriolar protein A (POC1A) effect in pan-cancer remains uncertain. The POC1A expression in normal and tumor tissues underwent analysis in this study utilizing data from the Genotype-Tissue Expression (GTEx) project and the Cancer Genome Atlas (TCGA) database. POC1A prognostic value and clinicopathological features were assessed utilizing the TCGA cohort. The relationship between immunological cell infiltration and POC1A of TCGA samples downloaded from TIMER2 and ImmuCellAI databases were observed. The relation between POC1A and immunological checkpoints genes, microsatellite instability (MSI) as well as tumor mutation burden (TMB) was also evaluated. Additionally, gene set enrichment analysis (GSEA) was utilized for exploring POC1A potential molecular mechanism in pan-cancer. In almost all 33 tumors, POCA1 showed a high expression. In most cases, high POC1A expression was linked significantly with a poor prognosis. Additionally, Tumor immune infiltration and tumors microenvironment were correlated with the expression of POC1A. In addition, TMB and MSI, as well as immune checkpoint genes in pan-cancer, were related to POC1A expression. In GSEA analysis, POC1A is implicated in cell cycle and immune-related pathways. These results might elucidate the crucial roles of POC1A in pan-cancer as a prognostic biomarker and immunotherapy target.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium.

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

The SARM1 TIR NADase: Mechanistic Similarities to Bacterial Phage Defense and Toxin-Antitoxin Systems.

The Toll/interleukin-1 receptor (TIR) domain is the signature signalling motif of innate immunity, with essential roles in innate immune signalling in bacteria, plants, and animals. TIR domains canonically function as scaffolds, with stimulus-dependent multimerization generating binding sites for signalling molecules such as kinases and ligases that activate downstream immune mechanisms. Recent studies have dramatically expanded our understanding of the TIR domain, demonstrating that the primordial function of the TIR domain is to metabolize NAD+. Mammalian SARM1, the central executioner of pathological axon degeneration, is the founding member of the TIR-domain class of NAD+ hydrolases. This unexpected NADase activity of TIR domains is evolutionarily conserved, with archaeal, bacterial, and plant TIR domains all sharing this catalytic function. Moreover, this enzymatic activity is essential for the innate immune function of these proteins. These evolutionary relationships suggest a link between SARM1 and ancient self-defense mechanisms that has only been strengthened by the recent discovery of the SARM1 activation mechanism which, we will argue, is strikingly similar to bacterial toxin-antitoxin systems. In this brief review we will describe the regulation and function of SARM1 in programmed axon self-destruction, and highlight the parallels between the SARM1 axon degeneration pathway and bacterial innate immune mechanisms.

Intrabody-Induced Cell Death by Targeting the T. brucei Cytoskeletal Protein TbBILBO1.

Trypanosoma brucei belongs to a genus of protists that cause life-threatening and economically important diseases of human and animal populations in Sub-Saharan Africa. T. brucei cells are covered in surface glycoproteins, some of which are used to escape the host immune system. Exo-/endocytotic trafficking of these and other molecules occurs via a single copy organelle called the flagellar pocket (FP). The FP is maintained and enclosed around the flagellum by the flagellar pocket collar (FPC). To date, the most important cytoskeletal component of the FPC is an essential calcium-binding, polymer-forming protein called TbBILBO1. In searching for novel tools to study this protein, we raised nanobodies (Nb) against purified, full-length TbBILBO1. Nanobodies were selected according to their binding properties to TbBILBO1, tested as immunofluorescence tools, and expressed as intrabodies (INb). One of them, Nb48, proved to be the most robust nanobody and intrabody. We further demonstrate that inducible, cytoplasmic expression of INb48 was lethal to these parasites, producing abnormal phenotypes resembling those of TbBILBO1 RNA interference (RNAi) knockdown. Our results validate the feasibility of generating functional single-domain antibody-derived intrabodies to target trypanosome cytoskeleton proteins. IMPORTANCE Trypanosoma brucei belongs to a group of important zoonotic parasites. We investigated how these organisms develop their cytoskeleton (the internal skeleton that controls cell shape) and focused on an essential protein (BILBO1) first described in T. brucei. To develop our analysis, we used purified BILBO1 protein to immunize an alpaca to make nanobodies (Nb). Nanobodies are derived from the antigen-binding portion of a novel antibody type found only in the camel and shark families of animals. Anti-BILBO1 nanobodies were obtained, and their encoding genes were inducibly expressed within the cytoplasm of T. brucei as intrabodies (INb). Importantly, INb48 expression rapidly killed parasites producing phenotypes normally observed after RNA knockdown, providing clear proof of principle. The importance of this study is derived from this novel approach, which can be used to study neglected and emerging pathogens as well as new model organisms, especially those that do not have the RNAi system.

Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

The receptor-type protein tyrosine phosphatase CD45 promotes onset and severity of IL-1ß-mediated autoinflammatory osteomyelitis.

A number of human autoinflammatory diseases manifest with severe inflammatory bone destruction. Mouse models of these diseases represent valuable tools that help us to understand molecular mechanisms triggering this bone autoinflammation. The Pstpip2cmo mouse strain is among the best characterized of these; it harbors a mutation resulting in the loss of adaptor protein PSTPIP2 and development of autoinflammatory osteomyelitis. In Pstpip2cmo mice, overproduction of interleukin-1ß (IL-1ß) and reactive oxygen species by neutrophil granulocytes leads to spontaneous inflammation of the bones and surrounding soft tissues. However, the upstream signaling events leading to this overproduction are poorly characterized. Here, we show that Pstpip2cmo mice deficient in major regulator of Src-family kinases (SFKs) receptor-type protein tyrosine phosphatase CD45 display delayed onset and lower severity of the disease, while the development of autoinflammation is not affected by deficiencies in Toll-like receptor signaling. Our data also show deregulation of pro-IL-1ß production by Pstpip2cmo neutrophils that are attenuated by CD45 deficiency. These data suggest a role for SFKs in autoinflammation. Together with previously published work on the involvement of protein tyrosine kinase spleen tyrosine kinase, they point to the role of receptors containing immunoreceptor tyrosine-based activation motifs, which after phosphorylation by SFKs recruit spleen tyrosine kinase for further signal propagation. We propose that this class of receptors triggers the events resulting in increased pro-IL-1ß synthesis and disease initiation and/or progression.

Examining the Effect of Kindlin-3 Binding Site Mutation on LFA-1-ICAM-1 Bonds by Force Measuring Optical Tweezers.

Integrins in effector T cells are crucial for cell adhesion and play a central role in cell-mediated immunity. Leukocyte adhesion deficiency (LAD) type III, a genetic condition that can cause death in early childhood, highlights the importance of integrin/kindlin interactions for immune system function. A TTT/AAA mutation in the cytoplasmic domain of the ß2 integrin significantly reduces kindlin-3 binding to the ß2 tail, abolishes leukocyte adhesion to intercellular adhesion molecule 1 (ICAM-1), and decreases T cell trafficking in vivo. However, how kindlin-3 affects integrin function in T cells remains incompletely understood. We present an examination of LFA-1/ICAM-1 bonds in both wild-type effector T cells and those with a kindlin-3 binding site mutation. Adhesion assays show that effector T cells carrying the kindlin-3 binding site mutation display significantly reduced adhesion to the integrin ligand ICAM-1. Using optical trapping, combined with back focal plane interferometry, we measured a bond rupture force of 17.85 ±0.63 pN at a force loading rate of 30.21 ± 4.35 pN/s, for single integrins expressed on wild-type cells. Interestingly, a significant drop in rupture force of bonds was found for TTT/AAA-mutant cells, with a measured rupture force of 10.08 ± 0.88pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the ß2-tail directly affects catch bond formation and bond strength of integrin-ligand bonds. As a consequence of this reduced binding, CD8+ T cell activation in vitro is also significantly reduced.

HSCT is effective in patients with PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome.

BACKGROUND: Proline-serine-threonine phosphatase-interacting protein 1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome is a novel genetic disorder, causing hypercalprotectinemia and hyperzincemia with inflammatory complications accompanied by cytopenia. Immunosuppressive and/or anticytokine therapy is of limited effect. OBJECTIVES: Because of cytokine production in nonhematopoietic tissues, the potential therapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) in autoinflammatory disorders, including PAMI syndrome, has remained uncertain. METHODS: Five patients with PAMI syndrome underwent allogeneic HSCT with myeloablative (4) or reduced-intensity (1) conditioning regimens. Lack of PAMI disease control served as indication for the HSCT in 4 patients and myelodysplastic syndrome development in 1. RESULTS: All 5 patients engrafted; however, 1 patient at day +13 developed hemophagocytic syndrome, followed by graft rejection at day +17. After 5.5 months, a second HSCT was performed from an alternative donor. A further patient at day +116 developed an intense inflammatory syndrome with significant serositis and severe mitral and aortic valve regurgitation, controlled with adalimumab, tacrolimus, and prednisone. No other noninfectious inflammatory episodes, or acute or chronic graft-versus-host disease, occurred in any patient. At the last follow-up (median, 2.2 years), all 5 patients have predominantly or complete donor chimerism and adequate immune recovery and are free of any PAMI symptoms. CONCLUSIONS: Allogeneic HSCT seems to be an effective option to cure cytopenia and severe autoinflammation in PAMI syndrome and may be a curative option for other proline-serine-threonine phosphatase-interacting protein 1-associated inflammatory disorders with poor therapeutic control.

Targeted MRI and chemotherapy of ovarian cancer with clinic available nano-drug based nanoprobe.

Cancer is the leading cause of death worldwide, and chemotherapy, as its main treatment, has side effects in clinical application due to lack of targeting selectivity to tumor tissues. Theranostic nanomaterials have shown wonderful functions for the diagnosis and therapy of disease benefitting from the controllability of nanomaterials. However, there is still little available for clinical transformation due to the uncertain biocompatibility. It is urgent to develop nanoprobes possessing bright transformation potentials. This study reports a facile biomineralization route to synthesize the theranostic nanoprobe using the clinic available nano-drug (trademark Abraxane). Further profiting from the binding ability of albumin to metal cations, we successfully prepared biocompatible nanoprobe, BSA-Gd2O3/PTX@Anti-HE4 mAb, for the targeted magnetic resonance imaging and chemotherapy of ovarian carcinoma. The obtained nanoprobe has the advantages of uniform particle size, good dispersibility and favorable stability. In vivo and in vitro experiment results showed that the nanoprobe can realize targeted magnetic resonance imaging and chemotherapy of ovarian carcinoma. Such a novel multifunctional nanoprobe based on clinic nano-drug might be promising for clinic transformation.

Interferon-Induced Protein 44 and Interferon-Induced Protein 44-Like Restrict Replication of Respiratory Syncytial Virus.

Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.

BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.

Molecular cloning, characterization and expression profiles of CD2AP in Nile tilapia (Oreochromis niloticus) responding to Streptococcus agalactiae infection and interaction with CD2 cytoplasmic segment.

The interaction between CD2-associated protein (CD2AP) and CD2 plays a vital role in lymphocyte adhesion and T cells activation in mammals. In this study, a CD2AP gene (GenBank accession number: MK579862; designated as On-CD2AP) was identified from tilapia (Oreochromis niloticus). Sequence analysis showed that On-CD2AP protein shares high similarity with mammals, including three Src homology 3 (SH3) domains, a section of poly proline motif and a coiled coil region. Transcription levels of On-CD2AP were detected in nine tissues of healthy Nile tilapia, and the highest expression levels were detected in the spleen and gill. On-CD2AP were significantly up-regulated in thymus, head kidney and brain after infected by Streptococcus agalactiae, as well as in head kidney leukocytes (HKLs) with LPS and LTA stimulation. Moreover, a section conserved pro-rich motif that are responsible for binding of CD2 to CD2AP were found in the CD2 cytoplasmic sequence of Nile tilapia (On-CD2C). A weak interaction between On-CD2AP and On-CD2C was proved by yeast two-hybrid assay. In addition, the recombinant proteins of CD2AP-His (rOn-CD2AP-His) and GST-CD2C (GST-rOn-CD2C) were obtained through prokaryotic expression system. His pull-down assay showed that rOn-CD2AP-His and GST-rOn-CD2C could bind to each other. These findings indicate that CD2AP is crucial in immune response during S.agalactiae infection, and the mechanism of interaction between CD2AP and CD2 is conservative in Nile tilapia.

S100A7/Ran-binding protein 9 coevolution in mammals.

S100A7 has been suggested to interact with Ran-binding protein 9. Both proteins are nowadays considered key effectors in immune response. Functional interaction between proteins is ensured by coevolution. The mechanisms of vertebrate coevolution between S100A7 and RanBP9 remain unclear. Several approaches for studying coevolution have been developed. Protein coevolution was inferred by calculating the linear correlation coefficients between inter-protein distance matrices using Mirrortree. We found an overall moderate correlation value (R = 0.53, p < 1e-06). Moreover, owing to the high conservation of RanBP9 protein among vertebrates, we chose to utilize a recent version of Blocks in Sequences (BIS2) algorithm implemented in BIS2Analyzer webserver. A coevolution cluster was identified between the two proteins (p < 8.10e-05). In conclusion, our coevolutionary analysis suggests that amino acid variations may modulate S100A7/RanBP9 interaction with potential pathogenic effects. Such findings could guide further analysis to better elucidate the function of S100A7 and RanBP9 and to design drugs targeting for these molecules in diseases.

CG-NAP/Kinase Interactions Fine-Tune T Cell Functions.

CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions. As a result, new binding partners of CG-NAP are continually being uncovered. Here, we review recent advances in CG-NAP research, focusing on its interactions with kinases in T cells with an emphasis on the possible role of this anchoring protein as a target for therapeutic intervention in immune-mediated diseases.

Cytoskeletal and extracellular matrix proteins resist the burning of bones.

Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.

Fugu, Takifugu ruberipes, mucus keratins act as defense molecules against fungi.

Keratin is a cytoskeletal protein that constitutes the intermediate filament. Its distribution is restricted to epithelial tissues in mammals, but is wider in fish. An interesting feature of fish keratin is that it is abundant in the cutaneous mucus. However, the biological function of keratin in the mucus has not been explored. In the present study, we hypothesized that mucus keratins of fugu Takifugu rubripes function as antimicrobial molecules. To verify this hypothesis, we first identified all of the keratins expressed in the epidermis and present in mucus. Five of 15 keratins including Tr-K4 expressed in the epidermis were identified in the mucus. Subsequently, we examined the interaction of keratin molecules present in fugu mucus with yeast. Affinity chromatography using yeast as a carrier and subsequent LC-MS/MS analysis revealed that three types of keratin were bound to the yeast. Furthermore, yeast incubated with fugu mucus was agglutinated, and this was inhibited by anti-recombinant Tr-K4 (rTr-K4) antibody. Immunohistochemical analysis also revealed that keratin was attached to the surface of agglutinated yeasts. These findings indicate that mucus keratin agglutinates yeast. Furthermore, we found insoluble clumps in fugu mucus, which were mainly comprised of keratin. After incubation of yeast with soluble mucus fraction, insoluble clumps incorporating yeast were formed. This observation suggests that fugu mucus keratin sequesters microbes into insoluble clumps, which are eventually eliminated from the mucus. Here, we present our finding of the novel function of keratin as a defense molecule in fish mucus.

WDR90 is a new component of the NLRC4 inflammasome involved in Salmonella Typhimurium resistance.

Inflammasomes are pivotal cytosolic molecular platforms involved in infection resistance. As multiprotein complexes, they consist of NOD-like receptors (NLRs), the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and the effector molecules caspase-1 and caspase-11, whose assembly and activation depends on homotypic interactions. Here we describe WD repeat containing protein 90 (WDR90) as a new inflammasome component. We found that zebrafish wdr90 is highly induced by guanylate binding protein 4 (Gbp4) independently of inflammasome activation and caspase-1 activity. This gene encodes an evolutionarily conserved protein with unknown functions that contains several WD40 domains, which are involved in coordinating multiprotein complex assembly. Functional studies in zebrafish larvae showed that forced expression of wdr90 increased caspase-1 activity and inflammasome-dependent resistance to Salmonella enterica serovar Typhimurium infection. Wdr90 acted upstream of zebrafish caspase a (Caspa), the functional homolog of mammalian caspase-1, and Asc. Reconstitution experiments of the human inflammasome in HEK293 cells demonstrated that WDR90 was able to physically interact with and to alter the cellular distribution of NLRC4, but not of NLRP3 and AIM2. These results highlight the complexity of the inflammasome and the interest of studying fish immunity to understand not only the evolution of the immune system but also human immunity.

Antibodies Used to Detect Glaucoma-Associated Myocilin: More or Less Than Meets the Eye?

Antibodies are key reagents used in vision research, indeed across biomedical research, but they often do not reveal the whole story about a sample. It is important for researchers to be aware of aspects of antibodies that may affect or limit data interpretation. Federal agencies now require funded grants to demonstrate how they will authenticate reagents used. There is also a push for recombinant antibodies, enabled by phage display technology awarded the 2018 Nobel Prize in Chemistry, which allow for thorough validation and a fixed DNA sequence. Here, we discuss how issues surrounding antibodies are pertinent to detecting myocilin, a protein found in trabecular meshwork and associated with a portion of hereditary glaucoma. Confirmation of myocilin expression in tissues and cell culture has been adopted as validation standard in trabecular meshwork research; thus, a discussion of antibody characteristics and fidelity is critical. Further, based on our basic structural understanding of myocilin architecture and its biophysical aggregation properties, we provide a wish list for the characteristics of next-generation antibody reagents for vision researchers. In the long term, well-characterized antibodies targeting myocilin will enable new insights into its function and involvement in glaucoma pathogenesis.