SummaryHypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.

SUMMARYExamination of embryonic myogenesis of two distinct, but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3 -/-) establishes for the first time that key elements of the pathology of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 1C (LGMD1c) originate in the disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdx mutants, which lack a functional form of dystrophin, than in cav-3 -/-mutants, which lack the Cav3 gene that encodes the protein caveolin-3; this finding is consistent with the milder phenotype of LGMD-1c, a condition caused by mutations in Cav3, and the earlier [embryonic day (E)9.5] expression of dystrophin. Myogenesis is severely disrupted in mdx embryos, which display developmental delays; myotube morphology and displacement defects; and aberrant stem cell behaviour. In addition, the caveolin-3 protein is elevated in mdx embryos. Both cav-3 -/-and mdx mutants (from E15.5 and E11.5, respectively) exhibit hyperproliferation and apoptosis of Myf5-positive embryonic myoblasts; attrition of Pax7-positive myoblasts in situ; and depletion of total Pax7 protein in late gestation. Furthermore, both cav-3 -/-and mdx mutants have cardiac defects. In cav-3 -/-mutants, there is a more restricted phenotype comprising hypaxial muscle defects, an excess of malformed hypertrophic myotubes, a twofold increase in myonuclei, and reduced fast myosin heavy chain (FMyHC) content. Several mdx mutant embryo pathologies, including myotube hypotrophy, reduced myotube numbers and increased FMyHC, have reciprocity with cav-3 -/-mutants. In double mutant (mdxcav-3) embryos that are deficient in dystrophin (mdx) and heterozygous for caveolin-3 (cav-3), whereby caveolin-3 is reduced to 50% of wild-type (WT) levels, these phenotypes are severely exacerbated: intercostal muscle fibre density is reduced by 71%, and Pax7-positive cells are depleted entirely from the lower limbs and severely attenuated elsewhere; these data suggest a compensatory rather than a contributory role for the elevated caveolin-3 levels that are found in mdx embryos. These data establish a key role for dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential for correct fibre-type specification and emergent stem cell function. These data plug a significant gap in the natural history of muscular dystrophy and will be invaluable in establishing an earlier diagnosis for DMD/LGMD and in designing earlier treatment protocols, leading to better clinical outcome for these patients.

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.

Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1 L1376P ), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.

Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging. Here we describe the discovery of a high-affinity (Kd = 10 nM) peptide macrocycle (M21) against human tumor necrosis factor-alpha (hTNFα), a key drug target in the treatment of inflammatory disorders, directly from diverse semi-synthetic phage peptide repertoires. The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNFα cytokine into dimers and monomers. A 2.9 Å crystal structure of the M21/hTNFα complex reveals the peptide bound to a hTNFα dimer at a normally buried epitope in the trimer interface overlapping the binding site of a previously discovered small molecule ligand (SPD304), which also induces TNF trimer dissociation and synergizes with M21 in the inhibition of TNFα cytotoxicity. The discovery of M21 underlines the potential of semi-synthetic bicyclic peptides as ligands for the discovery of cryptic epitopes, some of which are poorly accessible to antibodies.

Proteins mediate the bulk of biological activity and are powerfully assayed in the diagnosis of diseases. Protein detection relies largely on antibodies, which have significant technical limitations especially when immobilized on two-dimensional surfaces. Here, we report the integration of peptide aptamers with extended gate metal-oxide-semiconductor field-effect transistors (MOSFETs) to achieve label-free sub-picomolar target protein detection. Specifically, peptide aptamers that recognize highly related protein partners of the cyclin-dependent kinase (CDK) family are immobilized on the transistor gate to enable human CDK2 to be detected at 100 fM or 5 pg/mL, well within the clinically relevant range. The target specificity, ease of fabrication, and scalability of these FET arrays further demonstrate the potential application of the multiplexable field effect format to protein sensing.

Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein–protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in ‘loop 2’ allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

In the near future, personalised medicine and phase-0 trials will require that clinical practitioners move from the "one biomarker per disease" paradigm to the use of molecular signatures of disease for diagnosis and the prediction of a patient's response to treatment. These signatures will be composed of biomarkers specific to the disease, and will include over-expression of normal protein from a gene that does not carry a mutation; loss of expression of an essential protein; expression of a protein from a mutant gene; and metabolites whose levels are altered in disease. Surrogates for protein expression, such as alterations in the messenger RNA that encode for them, have already proved their value. The next challenge, then, in clinical biosensing is to enable the multiplexed detection of protein biomarkers, and perhaps the multiplexed detection of mixed biomarkers (metabolites, RNA and proteins) all in a single test. Given the plethora of available antibodies specific for biomarkers, why is this not already happening? We believe that the limitation lies in the nature of the antibody molecule itself, and especially the fact that antibodies have evolved to function in solution, while most diagnostic tests take place at a surface. We have accordingly turned to the design of alternative antibodies, and have identified a protein that appears to be unusually stable on surfaces. The new, non-antibody, scaffold protein is derived from human Stefin A, a natural inhibitor of the cathepsin family of proteases. We have engineered this protein so that it lacks natural binding partners, and introduced a series of new binding surfaces through randomisation or directed replacement of the surfaces used by Stefin A to bind to cathepsins. Our new probes show exquisite specificity and binding affinities comparable to antibodies, and can be used to probe biology in intact cells. More importantly, together and in collaboration with other groups in Chemistry or Engineering Departments, we have shown that these designer proteins can be used in optical detection of labelled target proteins from whole cell lysates in a highly multiplexed microarray format, as well as in label-free detection of unlabelled proteins using surface plasmon resonance, QCM, microcantilevers and using electrochemical assays on gold electrodes. We believe that the combination of optimised surface chemistry, robust and combinatorial designer biological probes and novel, robust and sensitive detection technologies will enable, in the near future, the introduction of multiplexed biomarker detection in the clinical setting, most likely in cancer where multiple biomarkers are known, but probes are still lacking.