Plexin-mediated neuronal development and neuroinflammatory responses in the nervous system.

Plexins are a large family of single-pass transmembrane proteins that mediate semaphorin signaling in multiple systems. Plexins were originally characterized for their role modulating cytoskeletal activity to regulate axon guidance during nervous system development. Thereafter, different semaphorin-plexin complexes were identified in the nervous system that have diverse functions in neurons, astrocytes, glia, oligodendrocytes, and brain derived-tumor cells, providing unexpected but meaningful insights into the biological activities of this protein family. Here, we review the overall structure and relevant downstream signaling cascades of plexins. We consider the current knowledge regarding the function of semaphorin-plexin cascades in the nervous system, including the most recent data regarding their roles in neuronal development, neuroinflammation, and glioma.

Hybrid in vitro/in silico analysis of low-affinity protein-protein interactions that regulate signal transduction by Sema6D.

Semaphorins constitute a large family of secreted and membrane-bound proteins that signal through cell-surface receptors, plexins. Semaphorins generally use low-affinity protein-protein interactions to bind with their specific plexin(s) and regulate distinct cellular processes such as neurogenesis, immune response, and organogenesis. Sema6D is a membrane-bound semaphorin that interacts with class A plexins. Sema6D exhibited differential binding affinities to class A plexins in prior cell-based assays, but the molecular mechanism underlying this selectivity is not well understood. Therefore, we performed hybrid in vitro/in silico analysis to examine the binding mode of Sema6D to class A plexins and to identify residues that give rise to the differential affinities and thus contribute to the selectivity within the same class of semaphorins. Our biophysical binding analysis indeed confirmed that Sema6D has a higher affinity for Plexin-A1 than for other class A plexins, consistent with the binding selectivity observed in the previous cell-based assays. Unexpectedly, our present crystallographic analysis of the Sema6D-Plexin-A1 complex showed that the pattern of polar interactions is not interaction-specific because it matches the pattern in the prior structure of the Sema6A-Plexin-A2 complex. Thus, we performed in silico alanine scanning analysis and discovered hotspot residues that selectively stabilized the Sema6D-Plexin-A1 pair via Van der Waals interactions. We then validated the contribution of these hotspot residues to the variation in binding affinity with biophysical binding analysis and molecular dynamics simulations on the mutants. Ultimately, our present results suggest that shape complementarity in the binding interfaces is a determinant for binding selectivity.

The involvement of semaphorin 7A in tumorigenic and immunoinflammatory regulation.

Semaphorins, a large group of highly conserved proteins, consist of eight subfamilies that are widely expressed in vertebrates, invertebrates, and viruses and exist in membrane-bound or secreted forms. First described as axon guidance cues during neurogenesis, semaphorins also perform physiological functions in other organ systems, such as bone homeostasis, immune response, and tumor progression. Semaphorin 7A (SEMA7A), also known as CDw108, is an immune semaphorin that modulates diverse immunoinflammatory processes, including immune cell interactions, inflammatory infiltration, and cytokine production. In addition, SEMA7A regulates the proliferation, migration, invasion, lymph formation, and angiogenesis of multiple types of tumor cells, and these effects are mediated by the interaction of SEMA7A with two specific receptors, PLXNC1 and integrins. Thus, SEMA7A is intimately related to the pathogenesis of multiple autoimmune and inflammation-related diseases and tumors. This review focuses on the role of SEMA7A in the pathogenesis of autoimmune disorders, inflammatory diseases, and tumors, as well as the underlying mechanisms. Furthermore, strategies targeting SEMA7A as a potential predictive, diagnostic, and therapeutic agent for these diseases are also addressed.

The Role of Semaphorins in Metabolic Disorders.

Semaphorins are a family originally identified as axonal guidance molecules. They are also involved in tumor growth, angiogenesis, immune regulation, as well as other biological and pathological processes. Recent studies have shown that semaphorins play a role in metabolic diseases including obesity, adipose inflammation, and diabetic complications, including diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic wound healing, and diabetic osteoporosis. Evidence provides mechanistic insights regarding the role of semaphorins in metabolic diseases by regulating adipogenesis, hypothalamic melanocortin circuit, immune responses, and angiogenesis. In this review, we summarize recent progress regarding the role of semaphorins in obesity, adipose inflammation, and diabetic complications.

Structural basis of semaphorin-plexin cis interaction.

Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

Cryo-EM structure of the PlexinC1/A39R complex reveals inter-domain interactions critical for ligand-induced activation.

Plexins are receptors for semaphorins that transduce signals for regulating neuronal development and other processes. Plexins are single-pass transmembrane proteins with multiple domains in both the extracellular and intracellular regions. Semaphorin activates plexin by binding to its extracellular N-terminal Sema domain, inducing the active dimer of the plexin intracellular region. The mechanism underlying this activation process of plexin is incompletely understood. We present cryo-electron microscopic structure of full-length human PlexinC1 in complex with the viral semaphorin mimic A39R. The structure shows that A39R induces a specific dimer of PlexinC1 where the membrane-proximal domains from the two PlexinC1 protomers are placed close to each other, poised to promote the active dimer of the intracellular region. This configuration is imposed by a distinct conformation of the PlexinC1 extracellular region, stabilized by inter-domain interactions among the Sema and membrane-proximal domains. Our mutational analyses support the critical role of this conformation in PlexinC1 activation.

Semaphorins in Angiogenesis and Autoimmune Diseases: Therapeutic Targets?

The axonal guidance molecules, semaphorins, have been described to function both physiologically and pathologically outside of the nervous system. In this review, we focus on the vertebrate semaphorins found in classes 3 through 7 and their roles in vascular development and autoimmune diseases. Recent studies indicate that while some of these vertebrate semaphorins promote angiogenesis, others have an angiostatic function. Since some semaphorins are also expressed by different immune cells and are known to modulate immune responses, they have been implicated in autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. We conclude this review by addressing strategies targeting semaphorins as potential therapeutic agents for angiogenesis and autoimmune diseases.

Cell guidance ligands, receptors and complexes - orchestrating signalling in time and space.

Members of four cell guidance molecule families (the netrins, slits, ephrins and semaphorins) interact with their cognate cell surface receptors to guide cells during development and maintain tissue homeostasis. Integrated structure and cell-based analyses are providing insight into the mechanisms by which these signalling systems can deliver myriad outcomes that require exquisite accuracy in timing and location. Here we review recent advances in our understanding of the roles of oligomeric states, auto-inhibition, signalling assembly size and composition in cell guidance cue function.

Identification of a novel heterozygous missense mutation of SEMA3E (c.1327G>A; p. Ala443Thr) in a labor induced fetus with CHARGE syndrome.

BACKGROUND: CHARGE syndrome is a complex multisystem genetic disease. We aimed to find the potential gene mutation in the labor induced fetus with CHARGE syndrome. METHODS: Genomic DNA was extracted from the fetal thigh muscle tissue and the peripheral blood of his parents. The resulting exomes were sequenced using whole exome sequencing (WES) followed by the selection of the candidate causative mutation genes. The deleteriousness of the identified variants was predicted. Analysis of multiple alignment of protein sequences and protein conserved domains was performed by online software. Finally, Sanger sequencing was applied for validation of the identified variants in the WES. RESULTS: After sequencing and bioinformatics filtering, a heterozygous missense mutation of SEMA3E (c.1327G>A; p. Ala443Thr) was found in the fetus, while the mutation was absent in his parents. Genotyping results showed that the mutation cosegregated fully with definite CHARGE phenotypes between the fetus and his parents. This change was located in the Sema superfamily and highly conserved across different species. Sanger validation result was consistent with the WES analysis. CONCLUSION: Our investigations suggested that the heterozygous missense mutation of SEMA3E (c.1327G>A; p. Ala443Thr) may be a potential causal variant in the fetus with CHARGE syndrome.

A trimetallic CuAuPd nanowire as a multifunctional nanocomposites applied to ultrasensitive electrochemical detection of Sema3E.

In this work, an innovative metal nanowire-based biosensor designed for the quantitative detection of semaphorin 3E (Sema 3E), a potential biomarker for several diseases such as atherosclerosis and systemic sclerosis, was proposed. For the biosensor fabrication, novel trimetallic CuAuPd nanowire networks (NNWs) were synthesized to utilize as a multifunctional substrate for electron transfer, antibody immobilization and signal amplification via catalyzing the decomposition of hydrogen peroxide. A facile one-step approach was employed at room temperature and atmospheric pressure to synthesize the CuAuPd NNWs, exhibiting advantages of high specific surface area, excellent electron transport property, superior catalytic property, and excellent biocompatibility. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) was examined to determine the successful fabrication process of the sensor, while the electrochemical method of amperometric i-t curve was used for the detection of target. The results demonstrated accepted stability, excellent selectivity, sensitivity and accuracy, which displayed a linear range of the analyte concentration that covered 100 fg mL-1 to 10 ng mL-1, with a low detection limit of approximately 1.5 fg mL-1 (S/N = 3), achieved under optimum conditions. This result suggests that the sensor could be applied to the serum samples for Sema 3E quantitation.

PKC induces release of a functional ectodomain of the guidance cue semaphorin6A.

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)-induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact-mediated functions, Sema6A may have regulated, long-range, forward-signaling capacity.

Neuroimmune semaphorins as costimulatory molecules and beyond.

Several neuronal guidance proteins, known as semaphorin molecules, function in the immune system. This dual tissue performance has led to them being defined as "neuroimmune semaphorins". They have been shown to regulate T cell activation by serving as costimulatory molecules. Similar to classical costimulatory molecules, neuroimmune semaphorins are either constitutively or inducibly expressed on immune cells. In contrast to the classical costimulatory molecule function, the action of neuroimmune semaphorins requires the presence of two signals, the first one provided by TCR/MHC engagement, and the second one provided by B7/CD28 interaction. Thus, neuroimmune semaphorins serve as a "signal three" for immune cell activation and regulate the overall intensity of immune response. The current knowledge on their structures, multiple receptors, specific cell/tissue/organ expression, and distinct functions in different diseases are summarized and discussed in this review.

Real-time QCM-D monitoring of endothelial cells and macrophages adhering and spreading to SEMA4D/heparin surfaces.

In vitro evaluation techniques that address cellular interactions under dynamic circumstances are required for the development of advanced biomaterials. In this study, quartz crystal microbalance and dissipation (QCM-D) provided a surface sensitive technique that enabled real-time monitoring of the adhesion and spreading response of endothelial cells (ECs) and macrophages (MAs) to soluble-semaphoring 4D (SEMA4D)/heparin modified substratum. The high heparin and low SEMA4D of substratum promoted ECs adhering and spreading. However, surfaces with high SEMA4D and low heparin enhanced MA adhering and spreading. Furthermore, DF plots of QCM-D indicated that the adhering and spreading of MAs were mainly mediated via the ligand-receptor interaction of SEMA4D-CD72, while both adhering and spreading of ECs were mainly mediated via heparin-induced integrin binding. This study suggests that QCM-D combined with other appropriate methods can be utilized to explore the mechanisms for cellular interaction with biomaterial surfaces.

Sema-1a Reverse Signaling Promotes Midline Crossing in Response to Secreted Semaphorins.

Commissural axons must cross the midline to form functional midline circuits. In the invertebrate nerve cord and vertebrate spinal cord, midline crossing is mediated in part by Netrin-dependent chemoattraction. Loss of crossing, however, is incomplete in mutants for Netrin or its receptor Frazzled/DCC, suggesting the existence of additional pathways. We identified the transmembrane Semaphorin, Sema-1a, as an important regulator of midline crossing in the Drosophila CNS. We show that in response to the secreted Semaphorins Sema-2a and Sema-2b, Sema-1a functions as a receptor to promote crossing independently of Netrin. In contrast to other examples of reverse signaling where Sema1a triggers repulsion through activation of Rho in response to Plexin binding, in commissural neurons Sema-1a acts independently of Plexins to inhibit Rho to promote attraction to the midline. These findings suggest that Sema-1a reverse signaling can elicit distinct axonal responses depending on differential engagement of distinct ligands and signaling effectors.

Variable expressivity and genetic heterogeneity involving DPT and SEMA3D genes in autosomal dominant familial Meniere's disease.

Autosomal dominant (AD) familial Meniere's disease (FMD) is a rare disorder involving the inner ear defined by sensorineural hearing loss, tinnitus and episodic vertigo. Here, we have identified two novel and rare heterozygous variants in the SEMA3D and DPT genes segregating with the complete phenotype that have variable expressivity in two pedigrees with AD-FMD. A detailed characterization of the phenotype within each family illustrates the clinical heterogeneity in the onset and progression of the disease. We also showed the expression of both genes in the human cochlea and performed in silico analyses of these variants. Three-dimensional protein modelling showed changes in the structure of the protein indicating potential physical interactions. These results confirm a genetic heterogeneity in FMD with incomplete penetrance and variable expressivity.

Recruitment of Tiam1 to Semaphorin 4D Activates Rac and Enhances Proliferation, Invasion, and Metastasis in Oral Squamous Cell Carcinoma.

The semaphorins and the plexins are a family of large, cysteine-rich proteins originally identified as regulators of axon growth and lymphocyte activation that are now known to provide motility and positional information for a number of cell and tissue types. For example, our group and others have shown that some malignancies over express Semaphorin 4D (S4D), which acts through its receptor Plexin-B1 (PB1) on endothelial cells to attract blood vessels from the surrounding stroma for the purpose of supporting tumor growth. While plexins are the known functional receptors for the semaphorins, there is evidence that transmembrane semaphorins may transmit a signal themselves through their short cytoplasmic tail, a phenomenon known as 'reverse signaling.' We used computational methods based upon correlated evolution of sequences of interacting proteins, mutational analysis and in vitro and in vivo measurements of tumor aggressiveness to show that when bound to PB1, transmembrane S4D associates with the Rac GTPase exchange factor T lymphoma invasion and metastasis (Tiam) 1, which activates Rac and promotes proliferation, invasion and metastasis in oral squamous cell carcinoma (OSCC) cells. These results suggest that not only can S4D production by tumor cells affect the microenvironment, but engagement of this semaphorin at the cell surface activates a reverse signaling mechanism that influences tumor aggressiveness in OSCC.

The regulation of Sema4D exodomain shedding by protein kinase A in platelets.

We have previously shown that Sema4D expressed on the platelet plasma membrane can be cleaved by the metalloprotease ADAM17, producing a 120-kDa exodomain fragment that retains biological activity and remnant fragments of 24-28 kDa that remain associated with the platelet membrane. This process is modulated by calmodulin. Here we investigated the potential role of protein kinase A (PKA) in these events. Using a pharmacological approach, we now show that inhibition of PKA by H89 is sufficient to induce Sema4D exodomain shedding, while activation of PKA inhibits agonist-initiated shedding. Studies on the regulatory mechanism show that the shedding induced by PKA inhibition is mediated by ADAM17, but, unlike agonist-induced shedding, does not involve the dissociation of calmodulin from the Sema4D cytoplasmic domain. In attempt to identify the cleavage sites for shedding, we found that ADAM17 mediates variable cleavages in the juxtamembrane region. Therefore, our data reveal a potential regulatory mechanism for the shedding of Sema4D in platelets.

Structure of the Plexin Ectodomain Bound by Semaphorin-Mimicking Antibodies.

Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these "semaphorin-mimicking" antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved "C" shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies.

Understanding cell signalling systems: paving the way for new therapies.

The cell-to-cell signalling mechanisms of multi-cellular organisms orchestrate human development during embryogenesis and control homeostasis in adult tissues. These are mechanisms vital to human health and perturbation of cell-to-cell signalling is a contributing factor in many pathologies including cancer. The semaphorin cell guidance cues and their cognate plexin receptors exemplify a cell-to-cell signalling system for which insights into mechanistic principles are emerging. X-ray crystallographic data from Diamond beam lines have enabled us to probe the inner workings of semaphorin-plexin signalling to atomic-level resolutions. Importantly, we can complement protein crystallographic results with biophysical and cellular studies to dovetail structural information with functional impact. The signature seven-bladed ß propeller 'sema' domain of the semaphorins forms a dimer; in contrast the equivalent domain in the plexins is monomeric. The generic architecture of a semaphorin-plexin complex is characterized by the dimeric semaphorin cross-linking two copies of the plexin receptor. For specific family members, the co-receptor neuropilin serves to bolster this architecture, but in all cases, the dimeric interaction lies at the core of the ligand receptor complex, providing the essential trigger for signalling.