Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.</p

Targeted modulation of tropoelastin structure and assembly

Tropoelastin, as the monomer unit of elastin, assembles into elastic fibers that impart strength and resilience to elastic tissues. Tropoelastin is also widely used to manufacture versatile materials with specific mechanical and biological properties. The assembly of tropoelastin into elastic fibers or biomaterials is crucially influenced by key submolecular regions and specific residues within these domains. In this work, we identify the functional contributions of two rarely occurring negatively charged residues, glutamate 345 in domain 19 and glutamate 414 in domain 21, in jointly maintaining the native conformation of the tropoelastin hinge, bridge and foot regions. Alanine substitution of E345 and/or E414 variably alters the positioning and interactive accessibility of these regions, as illustrated by nanostructural studies and detected by antibody and cell probes. These structural changes are associated with a lower propensity for monomer coacervation, cross-linking into morphologically and functionally atypical hydrogels, and markedly impaired and abnormal elastic fiber formation. Our work indicates the crucial significance of both E345 and E414 residues in modulating specific local structure and higher-order assembly of human tropoelastin

Subtle balance of tropoelastin molecular shape and flexibility regulates dynamics and hierarchical assembly

The assembly of the tropoelastin monomer into elastin is vital for conferring elasticity on blood vessels, skin, and lungs. Tropoelastin has dual needs for flexibility and structure in self-assembly. We explore the structure-dynamics-function interplay, consider the duality of molecular order and disorder, and identify equally significant functional contributions by local and global structures. To study these organizational stratifications, we perturb a key hinge region by expressing an exon that is universally spliced out in human tropoelastins. We find a herniated nanostructure with a displaced C terminus and explain by molecular modeling that flexible helices are replaced with substantial β sheets. We see atypical higher-order cross-linking and inefficient assembly into discontinuous, thick elastic fibers. We explain this dysfunction by correlating local and global structural effects with changes in the molecule’s assembly dynamics. This work has general implications for our understanding of elastomeric proteins, which balance disordered regions with defined structural modules at multiple scales for functional assembly.United States. Office of Naval Research (Presidential Early Career Award for Scientists and Engineers)National Institutes of Health (U.S.) (U01 EB014976

Deriving the ultrastructure of α-crustacyanin using lower-resolution structural and biophysical methods

The structure of α-crustacyanin has been determined to 30 Å resolution using negative-stain electron microscopy (EM) single-particle averaging and modelling with the β-crustacyanin dimer from the crystal structure (Protein Data Bank code 1gka), guided by PISA protein subunit interface calculations for 1gka, and compared with the protein arrangements observed in the crystal lattice of 1gka. This α-crustacyanin EM model has been checked against SAXS experimental data, including comparison with rigid-body models calculated from the SAXS data, and finally with analytical ultracentrifugation measurements

Structural analysis of X-Linked Retinoschisis mutations reveals distinct classes which differentially effect retinoschisin function

Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ~4.2Å resolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly

Autosomal Recessive Cutis Laxa 1C Mutations Disrupt the Structure and Interactions of Latent TGFβ Binding Protein-4

From Frontiers via Jisc Publications RouterHistory: collection 2021, received 2021-05-07, accepted 2021-07-23, epub 2021-09-03Publication status: PublishedLatent TGFβ binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFβ and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues

Cooperative folding of intrinsically disordered domains drives assembly of a strong elongated protein.

Bacteria exploit surface proteins to adhere to other bacteria, surfaces and host cells. Such proteins need to project away from the bacterial surface and resist significant mechanical forces. SasG is a protein that forms extended fibrils on the surface of Staphylococcus aureus and promotes host adherence and biofilm formation. Here we show that although monomeric and lacking covalent cross-links, SasG maintains a highly extended conformation in solution. This extension is mediated through obligate folding cooperativity of the intrinsically disordered E domains that couple non-adjacent G5 domains thermodynamically, forming interfaces that are more stable than the domains themselves. Thus, counterintuitively, the elongation of the protein appears to be dependent on the inherent instability of its domains. The remarkable mechanical strength of SasG arises from tandemly arrayed 'clamp' motifs within the folded domains. Our findings reveal an elegant minimal solution for the assembly of monomeric mechano-resistant tethers of variable length.This research was supported by Biotechnology and Biological Research Council Grants BB/J006459/1 (D.T.G. and J.C.), BB/J005029/1 (F.W. and J.R.P), BB/G019452/1 (O.E.F and D.J.B) and BB/G020671/1 (C.G.B. and J.R.P.). H.K.H.F. is supported by a studentship from a Wellcome Trust 4-year PhD programme (WT095024MA). C.M.J. is supported by the German Federal Ministry of Education and Research (BMBF), grant BIOSCAT (contract N° 05K12YE1). J.C. is a Wellcome Trust Senior Research Fellow (WT/095195). J.R.P holds a British Heart Foundation Senior Basic Science Fellowship (FS/12/36/29588). The authors acknowledge the use of EMBL SAXS beamline P12 at Petra-3 (DESY, Hamburg, Germany) and Maxim Petoukhov (EMBL) for providing a modified version of SASR EF. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). The authors would like to thank Diamond Light Source for beamtime (proposal mx-7864) and Johan Turkenburg and Sam Hart for assistance with crystal testing and data collection.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/ncomms827

Molecular mechanism of BMP signal control by Twisted gastrulation

Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1

Learning to drive in young adults with language impairment

Language impairment (LI) is a common developmental disorder which affects many aspects of young people’s functional skills and engagement with society. Little is known of early driving behaviour in those with this disability. This longitudinal study examines early driving experience in a sample of young adults with LI, compared with a sample of typically developing age-matched peers (AMPs). At age 24 years, significantly fewer participants with LI had acquired a driving licence. A crucial hurdle for those with LI appeared to be the Theory part of the (UK) test. Logistic regression analysis indicated that language ability and a measure of independence at age 17 contributed to the prediction of licence possession at age 24. There was no evidence of differences in traffic violations or accident rates between those with and without LI. There is little evidence that young people with LI are at greater risk on the roads than peers without LI, but some individuals with LI might benefit from support in the course of preparation for driving and in the driving test

Missense mutations that cause Van der Woude syndrome and popliteal pterygium syndrome affect the DNA-binding and transcriptional activation functions of IRF6

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype–phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype–phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity