Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Pineal progenitors originate from a non-neural territory limited by FGF signalling.

The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.

Common and distinct transcriptional signatures of mammalian embryonic lethality.

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.

Assessment of Proteolytic Activities in the Bone Marrow Microenvironment.

During cytokine- or chemotherapy-induced hematopoietic stem cell (HSC) mobilization, a highly proteolytic microenvironment can be observed in the bone marrow that has a strong influence on adhesive and chemotactic interactions of HSC with their niches. The increase of proteases during mobilization goes along with a decrease of endogenous protease inhibitors. Prominent members of the proteases involved in HSC mobilization belong to the families of matrix metalloproteinases and cathepsins, which are able to degrade chemokines/cytokines, extracellular matrix components, and membrane-bound adhesion receptors. To determine the functional activity of different proteolytic enzymes, zymographic analyses with different substrates and pH conditions can be employed. An involvement of cysteine cathepsins can be determined by the "active site labeling" technique using a modified inhibitor irreversibly binding to the active center of the enzymes. Intact or degraded chemokines and cytokines, which fall into the range between 1000 and 20,000 Da, can readily be detected by MALDI-TOF analysis. These three methods can help to detect proteolytic activities directly involved in the mobilization process.

Deep phenotyping in zebrafish reveals genetic and diet-induced adiposity changes that may inform disease risk.

The regional distribution of adipose tissues is implicated in a wide range of diseases. For example, proportional increases in visceral adipose tissue increase the risk for insulin resistance, diabetes, and CVD. Zebrafish offer a tractable model system by which to obtain unbiased and quantitative phenotypic information on regional adiposity, and deep phenotyping can explore complex disease-related adiposity traits. To facilitate deep phenotyping of zebrafish adiposity traits, we used pairwise correlations between 67 adiposity traits to generate stage-specific adiposity profiles that describe changing adiposity patterns and relationships during growth. Linear discriminant analysis classified individual fish according to an adiposity profile with 87.5% accuracy. Deep phenotyping of eight previously uncharacterized zebrafish mutants identified neuropilin 2b as a novel gene that alters adipose distribution. When we applied deep phenotyping to identify changes in adiposity during diet manipulations, zebrafish that underwent food restriction and refeeding had widespread adiposity changes when compared with continuously fed, equivalently sized control animals. In particular, internal adipose tissues (e.g., visceral adipose) exhibited a reduced capacity to replenish lipid following food restriction. Together, these results in zebrafish establish a new deep phenotyping technique as an unbiased and quantitative method to help uncover new relationships between genotype, diet, and adiposity.

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.

A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.

Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

Cloning of recombinant monoclonal antibodies from hybridomas in a single mammalian expression plasmid.

Antibodies are an integral part of biological and medical research. In addition, immunoglobulins are used in many diagnostic tests and are becoming increasingly important in the therapy of diseases. To express antibodies recombinantly, the immunoglobulin heavy and light chains are usually cloned into two different expression plasmids. Here, we describe a method for recombinant antibody expression from a single plasmid.

Development of an antigen microarray for high throughput monoclonal antibody selection.

Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host.

Myeloid cell receptor LRP1/CD91 regulates monocyte recruitment and angiogenesis in tumors.

Recruitment of monocytes into sites of inflammation is essential in the immune response. In cancer, recruited monocytes promote invasion, metastasis, and possibly angiogenesis. LDL receptor-related protein (LRP1) is an endocytic and cell-signaling receptor that regulates cell migration. In this study, we isografted PanO2 pancreatic carcinoma cells into mice in which LRP1 was deleted in myeloid lineage cells. Recruitment of monocytes into orthotopic and subcutaneous tumors was significantly increased in these mice, compared with control mice. LRP1-deficient bone marrow-derived macrophages (BMDM) expressed higher levels of multiple chemokines, including, most prominently, macrophage inflammatory protein-1α/CCL3, which is known to amplify inflammation. Increased levels of CCL3 were detected in LRP1-deficient tumor-associated macrophages (TAM), isolated from PanO2 tumors, and in RAW 264.7 macrophage-like cells in which LRP1 was silenced. LRP1-deficient BMDMs migrated more rapidly than LRP1-expressing cells in vitro. The difference in migration was reversed by CCL3-neutralizing antibody, by CCR5-neutralizing antibody, and by inhibiting NF-κB with JSH-23. Inhibiting NF-κB reversed the increase in CCL3 expression associated with LRP1 gene silencing in RAW 264.7 cells. Tumors formed in mice with LRP1-deficient myeloid cells showed increased angiogenesis. Although VEGF mRNA expression was not increased in LRP1-deficient TAMs, at the single-cell level, the increase in TAM density in tumors with LRP1-deficient myeloid cells may have allowed these TAMs to contribute an increased amount of VEGF to the tumor microenvironment. Our results show that macrophage density in tumors is correlated with cancer angiogenesis in a novel model system. Myeloid cell LRP1 may be an important regulator of cancer progression.

Processing of CXCL12 by different osteoblast-secreted cathepsins.

Hematopoietic stem and progenitor cells (HSPCs) are known to reside in specialized niches at the endosteum in the trabecular bone. Osteoblasts are the major cell type of the endosteal niche. It is well established that secreted proteases are involved in cytokine-induced mobilization processes that release stem cell from their niches. However, migratory processes such as the regular trafficking of HSPCs between their niches and the periphery are not fully understood. In the current study we analyzed whether osteoblast-secreted cysteine cathepsins are able to reduce the direct interaction of HSPCs with bone-forming osteoblasts. Isolated human osteoblasts were shown to secrete proteolytically active cysteine cathepsins, such as cathepsins B, K, L, and X. All of these cathepsins were able to digest, although with different efficacy, the chemokine CXCL12, which is known to be important for retaining HSPCs in their niches. Of the 4 identified cathepsins, only cathepsin X was able to reduce binding of HSPCs to osteoblasts. Interestingly, nonactivated pro-cathepsin X and mature cathepsin X did not interfere with HSPC-osteoblast interactions. Only pro-cathepsin X treated with dithiothreitol, which unfolds but does not lead to full maturation of cathepsin X, significantly reduced HSPC adhesion to osteoblasts. These observations argue for a role of the accessible cathepsin X prodomain in diminishing cell binding. Our findings strongly suggest that the cysteine cathepsins B, K, and L constitutively secreted by osteoblasts are part of the fine-tuned regulation of CXCL12 in the bone marrow, whereas pro-cathepsin X with its prodomain can affect HSPC trafficking in the niche.

A rapid and scalable method for selecting recombinant mouse monoclonal antibodies.

BACKGROUND: Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas. RESULTS: Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos. CONCLUSIONS: This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

Cathepsin X is secreted by human osteoblasts, digests CXCL-12 and impairs adhesion of hematopoietic stem and progenitor cells to osteoblasts.

BACKGROUND: Hematopoietic stem cells are retained within discrete bone marrow niches through the effects of cell adhesion molecules and chemokine gradients. However, a small proportion of hematopoietic stem cells can also be found trafficking in the peripheral blood. During induced stem cell mobilization a proteolytic microenvironment is generated, but whether proteases are also involved in physiological trafficking of hematopoietic stem cells is not known. In the present study we examined the expression, secretion and function of the cysteine protease cathepsin X by cells of the human bone marrow. DESIGN AND METHODS: Human osteoblasts, bone marrow stromal cells and hematopoietic stem and progenitor cells were analyzed for the secretion of cathepsin X by western blotting, active site labeling, immunofluorescence staining and activity assays. A possible involvement of cathepsin X in cell adhesion and CXCL-12-mediated cell migration was studied in functional assays. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) analysis revealed the digestion mechanism of CXCL-12 by cathepsin X. RESULTS: Osteoblasts and stromal cells secrete cathepsin X, whereas hematopoietic stem and progenitor cells do not. Using a cathepsin X-selective substrate, we detected the catalytic activity of cathepsin X in cell culture supernatants of osteoblasts. Activated cathepsin X is able to reduce cellular adhesive interactions between CD34(+) hematopoietic stem and progenitor cells and adherent osteoblasts. The chemokine CXCL-12, a highly potent chemoattractant for hematopoietic stem cells secreted by osteoblasts, is readily digested by cathepsin X. CONCLUSIONS: The exo-peptidase cathepsin X has been identified as a new member of the group of CXCL-12-degrading enzymes secreted by non-hematopoietic bone marrow cells. Functional data indicate that cathepsin X can influence hematopoietic stem and progenitor cell trafficking in the bone marrow.

Dopaminergic neuronal cluster size is determined during early forebrain patterning.

We have explored the effects of robust neural plate patterning signals, such as canonical Wnt, on the differentiation and configuration of neuronal subtypes in the zebrafish diencephalon at single-cell resolution. Surprisingly, perturbation of Wnt signaling did not have an overall effect on the specification of diencephalic fates, but selectively affected the number of dopaminergic (DA) neurons. We identified the DA progenitor zone in the diencephalic anlage of the neural plate using a two-photon-based uncaging method and showed that the number of non-DA neurons derived from this progenitor zone is not altered by Wnt attenuation. Using birthdating analysis, we determined the timing of the last cell division of DA progenitors and revealed that the change in DA cell number following Wnt inhibition is not due to changes in cell cycle exit kinetics. Conditional inhibition of Wnt and of cell proliferation demonstrated that Wnt restricts the number of DA progenitors during a window of plasticity, which occurs at primary neurogenesis. Finally, we demonstrated that Wnt8b is a modulator of DA cell number that acts through the Fz8a (Fzd8a) receptor and its downstream effector Lef1, and which requires the activity of the Fezl (Fezf2) transcription factor for this process. Our data show that the differential response of distinct neuronal populations to the Wnt signal is not a simple interpretation of their relative anteroposterior position. This study also shows, for the first time, that diencephalic DA population size is modulated inside the neural plate much earlier than expected, concomitant with Wnt-mediated regional patterning events.

Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon.

The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region - the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.

The prethalamus is established during gastrulation and influences diencephalic regionalization.

The vertebrate neural plate contains distinct domains of gene expression, prefiguring the future brain areas. In this study, we draw an extended expression map of the rostral neural plate that reveals discrete domains inside the presumptive posterior forebrain. We show, by fate mapping, that these well-defined cell populations will develop into specific diencephalic regions. To address whether these early subterritories are already committed to restricted identities, we began to analyse the consequences of ablation and transplantation of these specific cell populations. We found that precursors of the prethalamus are already specified and irreplaceable at late gastrula stage, because ablation of these cells results in loss of prethalamic markers. Moreover, when transplanted into the ectopic environment of the presumptive hindbrain, these cells still pursue their prethalamic differentiation program. Finally, transplantation of these precursors, in the rostral-most neural epithelium, induces changes in cell identity in the surrounding host forebrain. This cell-non-autonomous property led us to propose that these committed prethalamic precursors may play an instructive role in the regionalization of the developing diencephalon.

Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek.

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/beta catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/beta catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a beta catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the beta catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/beta catenin and Wnt/PCP pathways.

Mutations of the Drosophila zinc finger-encoding gene vielfältig impair mitotic cell divisions and cause improper chromosome segregation.

We describe the molecular characterization and function of vielfältig (vfl), a X-chromosomal gene that encodes a nuclear protein with six Krüppel-like C2H2 zinc finger motifs. vfl transcripts are maternally contributed and ubiquitously distributed in eggs and preblastoderm embryos, excluding the germline precursor cells. Zygotically, vfl is expressed strongly in the developing nervous system, the brain, and in other mitotically active tissues. Vfl protein shows dynamic subcellular patterns during the cell cycle. In interphase nuclei, Vfl is associated with chromatin, whereas during mitosis, Vfl separates from chromatin and becomes distributed in a granular pattern in the nucleoplasm. Functional gain-of-function and lack-of-function studies show that vfl activity is necessary for normal mitotic cell divisions. Loss of vfl activity disrupts the pattern of mitotic waves in preblastoderm embryos, elicits asynchronous DNA replication, and causes improper chromosome segregation during mitosis.

Gain-of-function screen for genes that affect Drosophila muscle pattern formation.

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.