Journal of Molecular Biology

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  • Gene-Specific Methylation Control of H3K9 and H3K36 on Neurotrophic BDNF versus Astroglial GFAP Genes by KDM4A/C Regulates Neural Stem Cell Differentiation
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20

    Author(s): Anna Cascante , Susanne Klum , Moumita Biswas , Beatriz Antolin-Fontes , Fanie Barnabé-Heider , Ola Hermanson

    Neural stem cell (NSC) state and fate depend on spatially and temporally synchronized transcriptional and epigenetic regulation of the expression of extrinsic signaling factors and intrinsic cell-specific genes, but the functional roles for chromatin-modifying enzymes in neural differentiation remain poorly understood. Here we show that the histone demethylases KDM4A (JMJD2A) and KDM4C (JMJD2C) are essential for proper differentiation of NSCs in vitro and in vivo. KDM4A/C were required for neuronal differentiation, survival and expression of the neurotrophic signaling factor BDNF in association with promoter H3K9 demethylation and RNA polymerase II recruitment. Unexpectedly, KDM4A/C were essential for selective H3K36 demethylation and loss of RNA polymerase II recruitment in transcribed regions of the astrocyte-characteristic gene GFAP, thereby in parallel repressing astrocytic differentiation by control of elongation. We propose that gene- and lysine-specific KDM4A/C-mediated control of histone methylation and thereby regulation of intrinsic factors and signaling factors such as BDNF provide a novel control mechanism of lineage decision.
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  • Additional Articles published in Volume 426
    [Oct 2014]

    Publication date: 9 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 20









    Categories: Journal Articles
  • The Microbiota, Chemical Symbiosis, and Human Disease
    [Oct 2014]

    Publication date: Available online 8 October 2014
    Source:Journal of Molecular Biology

    Author(s): Matthew R. Redinbo

    Our understanding of mammalian-microbial mutualism has expanded by combing microbial sequencing with evolving molecular and cellular methods, and unique model systems. Here, the recent literature linking the microbiota to diseases of three of the key mammalian mucosal epithelial compartments – nasal, lung and gastrointestinal (GI) tract – is reviewed with a focus on new knowledge about the taxa, species, proteins and chemistry that promote health and impact progression toward disease. The information presented is further organized by specific diseases now associated with the microbiota:, Staphylococcus aureus infection and rhinosinusitis in the nasal-sinus mucosa; cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), and asthma in the pulmonary tissues. For the vast and microbially dynamic GI compartment, several disorders are considered, including obesity, atherosclerosis, Crohn’s disease, ulcerative colitis, drug toxicity, and even autism. Our appreciation of the chemical symbiosis ongoing between human systems and the microbiota continues to grow, and suggest new opportunities for modulating this symbiosis using designed interventions.
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  • Structural and functional divergence of the aldolase fold in Toxoplasma gondii
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Michelle L. Tonkin , Andrei S. Halavaty , Raghavendran Ramaswamy , Jiapeng Ruan , Makoto Igarashi , Huân M. Ngô , Martin J. Boulanger

    Parasites of the phylum Apicomplexa are highly successful pathogens of humans and animals world-wide. As obligate intracellular parasites, they have significant energy requirements for invasion and gliding motility that are supplied by various metabolic pathways. Aldolases have emerged as key enzymes involved in these pathways, and all apicomplexans express one or both of fructose 1,6-bisphosphate (F16BP) aldolase and 2-deoxyribose 5-phosphate (dR5P) aldolase (DERA). Intriguingly, Toxoplasma gondii, a highly successful apicomplexan parasite, expresses F16BP aldolase (TgALD1), d5RP aldolase (TgDERA), and a divergent dR5P aldolase-like protein (TgDPA) exclusively in the latent bradyzoite stage. While the importance of TgALD1 in glycolysis is well established and TgDERA is also likely to be involved in parasite metabolism, the detailed function of TgDPA remains elusive. To gain mechanistic insight into the function of different T. gondii aldolases, we first determined the crystal structures of TgALD1 and TgDPA. Structural analysis revealed that both aldolases adopt a TIM barrel fold accessorized with divergent secondary structure elements. Structural comparison of TgALD1 and TgDPA with members of their respective enzyme families revealed that while the active site residues are conserved in TgALD1, key catalytic residues are absent in TgDPA. Consistent with this observation, biochemical assays showed that while TgALD1 was active on F16BP, TgDPA was inactive on dR5P. Intriguingly, both aldolases are competent to bind polymerized actin in vitro. Altogether, structural and biochemical analyses of T. gondii aldolase and aldolase-like proteins reveal diverse functionalization of the classic TIM barrel aldolase fold.
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  • Chromatin driven behavior of topologically associating domains
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Filippo Ciabrelli , Giacomo Cavalli

    Metazoan genomes are highly organized inside the cell nucleus. Topologically Associating Domains (TADs) represent the building blocks of genome organization, but their linear modularity does not explain alone their spatial organization. Indeed, the chromatin type adorning a TAD can shape its structure and drives its nuclear positioning and its function. Genome-wide association studies revealed mainly four chromatin types: active chromatin, Polycomb-repressed chromatin, null chromatin and constitutive heterochromatin. In this review we will describe the main three-dimensional features of each chromatin type and finally their relationships with TAD organization and epigenetic memory
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  • Cytoskeletal control of nuclear morphology and chromatin organization
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Nisha M. Ramdas , G.V. Shivashankar

    The nucleus is sculpted towards various morphologies during cellular differentiation and development. Alterations in nuclear shape often result in changes to chromatin organization and genome function. This is thought to be reflective of its role as a cellular mechanotransducer. Recent evidence has highlighted the importance of cytoskeletal organization in defining how nuclear morphology regulates chromatin dynamics. However the mechanisms underlying cytoskeletal control of chromatin remodeling are not well understood. We demonstrate here, the differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNAi knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in baso-lateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. Cytoskeleton reorganization leads to nuclear lamina deformation which influences heterochromatin localization and core histone protein mobility. We also show that modulations in actin-microtubule assembly results in differential gene expression patterns. Taken together, we suggest that perinuclear actin and baso-lateral microtubule organization exerts mechanical control on nuclear morphology and chromatin dynamics.
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  • Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Yun Peng , Jun Lu , Joyce J.W. Wong , Ross A. Edwards , Laura S. Frost , J.N. Mark Glover

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates a pair of TraM tetramers cooperatively binds an underwound sbmA site that contains 12 base pairs/turn. The sbmA is composed of 4 copies of a 5 base pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5 base pair sequence motif. Specificity is also dictated by the positioning of 2 base pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and motifs 3 and 4, whereas in pED208 sbmA there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA, in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.
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  • Precise and efficient antibody epitope determination through library design, yeast display and next generation sequencing
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Thomas Van Blarcom , Andrea Rossi , Davide Foletti , Purnima Sundar , Steven Pitts , Christine Bee , Jody Melton Witt , Zea Melton , Adela Hasa-Moreno , Lee Shaughnessy , Dilduz Telman , Lora Zhao , Wai Ling Cheung , Jan Berka , Wenwu Zhai , Pavel Strop , Javier Chaparro-Riggers , David L. Shelton , Jaume Pons , Arvind Rajpal

    The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies which neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.
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  • Structural and functional mechanisms of CRAC channel regulation
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Ann Hye-Ryong Shim , Leidamarie Tirado-Lee , Murali Prakriya

    In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. The physiological importance of CRAC channels for human health is underscored by studies indicating that mutations in CRAC channel genes produce a spectrum of devastating diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Moreover, from a basic science perspective, CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties and therefore serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Studies in the last two decades have revealed the cellular and molecular choreography of the CRAC channel activation process, and it is now established that opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensors, STIM1 and STIM2. In this review, we summarize the functional and structural mechanisms of CRAC channel regulation, focusing on recent advances in our understanding of the conformational and structural dynamics of CRAC channel gating.
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  • Structural determinants in prion protein folding and stability
    [Oct 2014]

    Publication date: Available online 2 October 2014
    Source:Journal of Molecular Biology

    Author(s): Federico Benetti , Xevi Biarnés , Francesco Attanasio , Gabriele Giachin , Enrico Rizzarelli , Giuseppe Legname

    Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein (PrPC). Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrPC is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies (TSE), respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated: (i) the essential role of the octapeptide region in prion protein folding and stability; (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species; (iii) the role of the disulfide bridge in prion protein folding.
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  • Mechanisms of integral membrane protein insertion and folding
    [Oct 2014]

    Publication date: Available online 30 September 2014
    Source:Journal of Molecular Biology

    Author(s): Florian Cymer , Gunnar von Heijne , Stephen H. White

    The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, and the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of membrane protein insertion and the folding of polytopic membrane proteins in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure, it is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion.
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  • First Experimental Evidence for the Presence of a CRISPR Toxin in Sulfolobus
    [Oct 2014]

    Publication date: Available online 30 September 2014
    Source:Journal of Molecular Biology

    Author(s): Fei He , Lanming Chen , Xu Peng

    Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.
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  • A highly conserved region essential for NMD in the Upf2 N-terminal domain
    [Oct 2014]

    Publication date: Available online 30 September 2014
    Source:Journal of Molecular Biology

    Author(s): Zaineb Fourati , Bijoyita Roy , Claudia Millan , Pierre-Damien Coureux , Stéphanie Kervestin , Herman van Tilbeurgh , Feng He , Isabel Usón , Allan Jacobson , Marc Graille

    Upf1, Upf2, and Upf3 are the principal regulators of Nonsense-mediated mRNA Decay (NMD), a cytoplasmic surveillance pathway that accelerates the degradation of mRNAs undergoing premature translation termination. These three proteins interact with each other, the ribosome, the translation termination machinery, and multiple mRNA decay factors, but the precise mechanism allowing the selective detection and degradation of nonsense-containing transcripts remains elusive. Here we have determined the crystal structure of the N-terminal mIF4G domain from Saccharomyces cerevisiae Upf2 and identified a highly conserved region in this domain that is essential for NMD and independent of Upf2’s binding sites for Upf1 and Upf3. Mutations within this conserved region not only inactivate NMD, but also disrupt Upf2 binding to specific proteins, including Dbp6, a DEAD-box helicase. Although current models indicate that Upf2 functions principally as an activator of Upf1 and a bridge between Upf1 and Upf3, our data suggest that it may also serve as a platform for the association of additional factors that play roles in premature translation termination and NMD.
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  • Heparan sulphate saccharides modify focal adhesions: Implication in mucopolysaccharidosis neuropathophysiology
    [Oct 2014]

    Publication date: Available online 28 September 2014
    Source:Journal of Molecular Biology

    Author(s): Julie Bruyère , Elise Roy , Jérôme Ausseil , Thomas Lemonnier , Guillaume Teyre , Delphine Bohl , Sandrine Etienne-Manneville , Hugues Lortat-Jacob , Jean Michel Heard , Sandrine Vitry

    Mucopolysaccharidoses type III (MPSIII, Sanfilippo syndrome) are genetic diseases due to deficient heparan sulphate saccharide digestion by lysosomal exoglycanases. Progressive accumulation of undigested saccharides causes early onset behavioural and cognitive symptoms. The precise role of these saccharides in the pathophysiological cascade is still unclear. We showed that exposure of wild type neural cells to exogenous soluble heparan sulphate fragments of at least eight saccharides activated integrin-based focal adhesions, which attach cells to the extracellular matrix. Focal adhesions were constitutively activated in MPSIII type B astrocytes or neural stem cells unless undigested saccharides were cleared by exogenous supply of the missing exoglycanase. Defective cell polarisation and oriented migration in response to focal extracellular stimuli in affected cells suggest improper sensing of the environment. We consistently observed abnormal organisation of the rostral migratory stream in the brain of adult mice with MPSIII type B. These results suggest that cell polarisation and oriented migration defects participate to the neurological disorders associated with Sanfilippo syndrome.
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  • Editorial Board
    [Oct 2014]

    Publication date: 23 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 19









    Categories: Journal Articles
  • Contents List
    [Oct 2014]

    Publication date: 23 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 19









    Categories: Journal Articles
  • Flipping through the Genetic Code: New Developments in Discrimination between Cognate and Near-Cognate tRNAs and the Effect of Antibiotics
    [Oct 2014]

    Publication date: 23 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 19

    Author(s): Karissa Y. Sanbonmatsu







    Categories: Journal Articles
  • Flipping of the Ribosomal A-Site Adenines Provides a Basis for tRNA Selection
    [Oct 2014]

    Publication date: 23 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 19

    Author(s): Xiancheng Zeng , Jeetender Chugh , Anette Casiano-Negroni , Hashim M. Al-Hashimi , Charles L. Brooks III

    Ribosomes control the missense error rate of ~10−4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near-cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA–tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA.
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  • A Similar In Vitro and In Cell Lysate Folding Intermediate for the FF Domain
    [Oct 2014]

    Publication date: 23 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 19

    Author(s): Michael P. Latham , Lewis E. Kay

    Understanding the mechanisms by which proteins fold into their three-dimensional structures, including a description of the intermediates that are formed during the folding process, remains a goal of protein science. Most studies are performed under carefully controlled conditions in which the folding reaction is monitored in a buffer solution that is far from the natural milieu of the cell. Here, we have used 13C and 1H relaxation dispersion NMR spectroscopy to study folding of the FF domain in both Escherichia coli and Saccharomyces cerevisiae cellular lysates. We find that a conformationally excited state is populated in both lysates, which is very similar in structure to a folding intermediate observed in previous studies in buffer, with the kinetics and thermodynamics of the interconversion between native and intermediate conformers somewhat changed. The results point to the importance of extending folding studies beyond the test tube yet emphasize that insights can be obtained through careful experiments recorded in controlled buffer solutions.
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  • Molecular Analysis of Two Novel Missense Mutations in the GDF5 Proregion That Reduce Protein Activity and Are Associated with Brachydactyly Type C
    [Oct 2014]

    Publication date: 23 September 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 19

    Author(s): Katja Stange , Tino Thieme , Karen Hertel , Silke Kuhfahl , Andreas R. Janecke , Hildegunde Piza-Katzer , Maila Penttinen , Marja Hietala , Katarina Dathe , Stefan Mundlos , Elisabeth Schwarz , Petra Seemann

    Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5T201P and proGDF5L263P revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.
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