Triple Helical Domain Research Articles

An outcome-defining role for the triple-helical domain in regulating collagen-I assembly

Collagens are the foundational component of diverse tissues, including skin, bone, cartilage, and basement membranes, and are the most abundant protein class in animals. The fibrillar collagens are large, complex, multidomain proteins, all containing the characteristic triple helix motif. The most prevalent collagens are heterotrimeric, meaning that cells express at least two distinctive procollagen polypeptides that must assemble into specific heterotrimer compositions. The molecular mechanisms ensuring correct heterotrimeric assemblies are poorly understood - even for the most common collagen, type-I. The longstanding paradigm is that assembly is controlled entirely by the ~30 kDa globular C-propeptide (C-Pro) domain. Still, this dominating model for procollagen assembly has left many questions unanswered. Here, we show that the C-Pro paradigm is incomplete. In addition to the critical role of the C-Pro domain in templating assembly, we find that the amino acid sequence near the C terminus of procollagen's triple-helical domain plays an essential role in defining procollagen assembly outcomes. These sequences near the C terminus of the triple-helical domain encode conformationally stabilizing features that ensure only desirable C-Pro-mediated trimeric templates are committed to irreversible triple-helix folding. Incorrect C-Pro trimer assemblies avoid commitment to triple-helix formation thanks to destabilizing features in the amino acid sequences of their triple helix. Incorrect C-Pro assemblies are consequently able to dissociate and search for new binding partners. These findings provide a distinctive perspective on the mechanism of procollagen assembly, revealing the molecular basis by which incorrect homotrimer assemblies are avoided and setting the stage for a deeper understanding of the biogenesis of this ubiquitous protein.

Phenotype-genotype spectrum of a cohort of congenital muscular dystrophies: a single-centre experience from India.

Congenital Muscular Dystrophies (CMD) are phenotypically and genotypically heterogenous disorders with a prevalence of 0.68 to 2.5/100,000, contributing to significant morbidity and mortality. We aimed to study the phenotype-genotype spectrum of genetically confirmed cases of CMD. This was retrospective & descriptive study done at a quaternary care referral centre in south India. Genetically confirmed cases of CMDs seen between 2010 to 2020 were recruited. Detailed clinical history, including pedigree, MRI brain/muscle, next generation sequencing results of 61 CMD cases were collected. Collagen VI-related dystrophy (COL6-RD) (36%) was the most common subtype with variants frequently seen in COL6A1 gene. Other CMDs identified were LAMA2-RD (26%), alpha-dystroglycan-RD (19%), LMNA-RD (8%), CHKB-RD (7%) and SEPN1-RD (3%). Similar to previous cohorts, overall, missense variants were common in COL-6 RD. Variants in triple helical domain (THD) of COL6-RD were seen in 11/22 patients, 5 of whom were ambulatory contrary to previous literature citing severe disease with these variants. However, our follow-up period was shorter. In the LAMA2-RD, 2/16 patients were ambulatory & all 16 carried truncating variants. Among dystroglycanopathies, FKRP-RD was the commonest. Milder phenotype of FKRP- RD was observed with variant c.1343C > T, which was also a recurrent variant in our cohort. p.Arg249Trp variant in LMNA-CMD associated with early loss of ambulation was also identified in 1/5 of our patients who expired at age 2.8years. The current retrospective series provides detailed clinical features and mutation patterns of genetically confirmed cases of CMD from a single center in India.

Exploring the Fibrous Nature of Single-Stranded DNA-Collagen Complexes: Nanostructural Observations and Physicochemical Insights.

Nucleic acid-collagen complexes (NACCs) are a self-assembled biomimetic fibrillary platform arising from the spontaneous complexation of single-stranded DNA (ssDNA) oligonucleotides and collagen. NACCs merge the extracellular matrix functionality of collagen with the tunable bioactivity of ssDNA as aptamers for broad biomedical applications. We hypothesize that NACCs offer a hierarchical architecture across multiple length scales that significantly varies compared to native collagen. We investigate this using atomic force microscopy and electron microscopy (transmission electron microscopy and cryogenic electron microscopy). Results demonstrate key topographical differences induced by adding ssDNA oligonucleotides to collagen type I. NACCs form a dense network of intertwined collagen fiber bundles in the microscale and nanoscale while retaining their characteristic D-band periodicities (∼67 nm). Additionally, our exploration of thermodynamic parameters governing the interaction indicates an entropically favorable NACC formation driven by ssDNA. Thermal analysis demonstrates the preservation of collagen's triple helical domains and a more stabilized polypeptide structure at higher temperatures than native collagen. These findings offer important insights into our understanding of the ssDNA-induced complexation of collagen toward the further establishment of structure-property relationships in NACCs and their future development into practical biomaterials. They also provide pathways for manipulating and enhancing collagenous matrices' properties without requiring complex chemical modifications or fabrication procedures.

Self-Assembling Triple-Helix Recombinant Collagen Hydrogel Enriched with Tyrosine.

The self-assembly of collagen within the human body creates a complex 3D fibrous network, providing structural integrity and mechanical strength to connective tissues. Recombinant collagen plays a pivotal role in the realm of biomimetic natural collagen. However, almost all of the reported recombinant collagens lack the capability of self-assembly, severely hindering their application in tissue engineering and regenerative medicine. Herein, we have for the first time constructed a series of self-assembling tyrosine-rich triple helix recombinant collagens, mimicking the structure and functionality of natural collagen. The recombinant collagen consists of a central triple-helical domain characterized by the (Gly-Xaa-Yaa)n sequence, along with N-terminal and C-terminal domains featuring the GYY sequence. The introduction of GYY has a negligible impact on the stability of the triple-helical structure of recombinant collagen while simultaneously promoting its self-assembly into fibers. In the presence of [Ru(bpy)3]Cl2 and APS as catalysts, tyrosine residues in the recombinant collagen undergo covalent cross-linking, resulting in a hydrogel with exceptional mechanical properties. The recombinant collagen hydrogel exhibits outstanding biocompatibility and bioactivity, significantly enhancing the proliferation, adhesion, migration, and differentiation of HFF-1 cells. This innovative self-assembled triple-helix recombinant collagen demonstrates significant potential in the fields of tissue engineering and medical materials.

Excited States of apo-Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm+) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm+ from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm+. We studied the binding of Gdm+ to the AD of the guanidine (ykkC)-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm+ binding. We show that Gdm+ binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm+ binding propensity, making the Gdm+ binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo-AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

Collagen VI-related myopathies: clinical variability, phenotype-genotype correlation and exploratory transcriptome study

Collagen VI-related myopathies are a group of disorders that cause muscle weakness and joint contractures with significant variability in disease severity among patients. Here we report the clinical and genetic characteristics of 13 Chinese patients. Detailed histological, radiological and muscle transcriptomic evaluations were also conducted for selected representative patients. Across the cohort, fifteen putative disease causal variants were identified in three genes encoding collagen VI subunits, COL6A1 (n=6), COL6A2 (n=5), and COL6A3 (n=4). Most of these variants (12/15, 80%) were dominant negative and occurred at the triple helical domain. The rest (3/15, 20%) were located at the C-terminus. Two previously unreported variants, an in-frame mutation (COL6A1:c.1084_1092del) and a missense mutation (COL6A2:c.811G>C), were also noted. The transcriptome data from the muscle biopsies of two patients in the study with dominant negative mutations [COL6A2:c.811G>C and COL6A1:c.930+189C>T] supports the accepted aetiology of Collagen VI myopathy as dysfunction of the extracellular matrix. It also suggests there are perturbations to skeletal muscle differentiation and skeletal system development. It should be noted that although the phenotypes of patients can be mostly explained by the position and dominant-negative effect of the variants, exceptions and variability still exist and have to be reckoned with. This study provides valuable data explaining the varying severity of phenotypes among ethnically Chinese patients.

Involvement of MMP-9 in collagen degradation of sea bass (Lateolabrax japonicus): Cloning, expression, and characterization.

Disintegration of intramuscular connective tissue is responsible for postmortem tenderization of fish muscles during chilled storage. Matrix metalloproteinase-9 (MMP-9) was reported to be involved in this process, whereas the mechanism has not been revealed. In the present study, purified type I and V collagens from the connective tissues of sea bass (Lateolabrax japonicus) muscles were first prepared. These two kinds of collagens comprise three polypeptide chains (α), forming a typical triple-helical domain as determined by circular dichroism. The complete coding region of MMP-9 containing an open reading frame of 2070bp encoding 689 amino acid residues was then cloned. The recombinant MMP-9 catalytic domain (rcMMP-9) was expressed in Escherichia coli and exhibited high hydrolyzing activity toward gelatin. Besides, rcMMP-9 was effective in degrading type V collagen rather than type I collagen at 4°C. The enzymatic activity of rcMMP-9 was highly pH-dependent, and its enzymatic activity under neutral and basic conditions was higher than that under acidic conditions. Metal ion Ca2+ was necessary for the maintenance of rcMMP-9 activity, whereas Zn2+ inhibited its activity. Our present study indicated that MMP-9 is responsible for the disintegration of intramuscular connective tissues by cleaving type V collagen during postmortem tenderization of fish muscle. PRACTICAL APPLICATION: Elucidation the involvement of MMP-9 in collagen degradation will deliver a reference for the prevention of muscular protein decomposition during chilled storage of fish fillets.

Comprehensive genetic screening for vascular Ehlers-Danlos syndrome through an amplification-based next-generation sequencing system.

Vascular Ehlers-Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys-Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.

COL1A1 novel splice variant in osteogenesis imperfecta and splicing variants review: A case report

Osteogenesis imperfecta (OI) is a rare heterogeneous genetic disorder commonly autosomal dominant with variants in the COL1A1 and COL1A2 genes. It is characterized by bone fragility and deformity, recurrent fractures, blue sclera, dentinogenesis imperfecta, short stature, and progressive deafness. We present a novel splicing mutation in the COL1A1 gene (c.2398-1G > C) in a 6-year-old Ecuadorian girl with fractures after light pressure and blue sclera. We identified the pathogenic variant, performed a literature review of splice variants, and recognized their location in the COL1A1 functional domains. We describe the first clinical description of a patient with OI type 1 caused by a splice variant in intron 34 of COL1A1 gene and identify that most of them are localized in the triple-helical region domain. We suggest that the splice variant in signal peptide, von Willebrand factor type C, and nonhelical regions maintain their functionality or that individuals affected with severe cases die early in development and are not reported.

Atypical variants in COL1A1 and COL3A1 associated with classical and vascular Ehlers-Danlos syndrome overlap phenotypes: expanding the clinical phenotype based on additional case reports.

The vast majority of reported (likely) pathogenic missense variants in the genes coding for the fibrillar collagens leads to the substitution of one of the obligatory glycine residues in the Gly-Xaa-Yaa repeat sequence of the triple helical domain. Their phenotypic consequences and deleterious effects have been well-documented. However, with increasing access to molecular diagnostic testing based on next-generation sequencing techniques, such as sequencing of multi-gene panels and whole-exome sequencing, non-glycine substitutions are more frequently identified in individuals suspected to have a heritable collagen disorder, but their pathogenic effect is often difficult to predict.Some specific non-glycine substitutions in the proα1(I)- (p.(Arg312Cys)) and proα1(III)- (glutamic acid to lysine at different positions) collagen chain have been identified in a number of individuals presenting a phenotype showing features of both classical and vascular Ehlers-Danlos syndrome. The number of reported individuals with these defects is currently very low, and several of these non-glycine substitutions had initially been categorised as variants of unknown significance (VUS), complicating early diagnosis, accurate counselling, management guidelines, and correct classification. This collaborative study reports on the phenotype of 22 and 7 individuals harbouring these rare variants in COL1A1 and COL3A1, respectively, expanding our knowledge on clinical presentation, phenotypic variability, and natural history, and informing on the risk for potentially life-threatening events, such as vascular, gastro-intestinal, and pregnancy-related complications.

Caffey disease is associated with distinct arginine to cysteine substitutions in the proα1(I) chain of type I procollagen

Infantile Caffey disease is a rare disorder characterized by acute inflammation with subperiosteal new bone formation, associated with fever, pain, and swelling of the overlying soft tissue. Symptoms arise within the first weeks after birth and spontaneously resolve before the age of two years. Many, but not all, affected individuals carry the heterozygous pathogenic COL1A1 variant (c.3040C>T, p.(Arg1014Cys)). We sequenced COL1A1 in 28 families with a suspicion of Caffey disease and performed ultrastructural, immunocytochemical, and biochemical collagen studies on patient skin biopsies. We identified the p.(Arg1014Cys) variant in 23 families and discovered a novel heterozygous pathogenic COL1A1 variant (c.2752C>T, p.(Arg918Cys)) in five. Both arginine to cysteine substitutions are located in the triple helical domain of the proα1(I) procollagen chain. Dermal fibroblasts (one patient with p.(Arg1014Cys) and one with p.(Arg918Cys)) produced molecules with disulfide-linked proα1(I) chains, which were secreted only with p.(Arg1014Cys). No intracellular accumulation of type I procollagen was detected. The dermis revealed mild ultrastructural abnormalities in collagen fibril diameter and packing. The discovery of this novel pathogenic variant expands the limited spectrum of arginine to cysteine substitutions in type I procollagen. Furthermore, it confirms allelic heterogeneity in Caffey disease and impacts its molecular confirmation.

Progress in the detection and quantification of collagens: a review

Collagens are an important and ubiquitous family of proteins. They have many functions in the human body and similarly have found numerous, potent applications in various industries including the manufacture of biomaterials. The ever-increasing demand for collagen has made necessary the exploration of alternative sources such as bacterial collagen-like proteins which have a triple-helical domain of Gly-X-Y amino acid repeats. Detection and quantification of native collagens have been well-established. However, collagen-like proteins differ in their composition and do not have the unique abundance of hydroxyproline and hydroxylysine found in vertebrate collagens. Thus, this poses a problem in the detection and quantification of collagen-like proteins. This paper evaluates reports on the detection and quantification of collagens and collagen-like proteins. A systematic search of the PubMed database was conducted in May 2021, to which five additional papers were added. The 310 unique search results were then subjected to a screening and elimination process, at the end of which 22 papers were included in the study. The findings were summarized and presented in a table that highlights progress in this field. While novel methods have been developed for the detection and quantitation of collagens in general, mainly using enzyme digestion, hybridization, and fluorescence, there is a need for a rapid, one-step method that selectively and sensitively detects and quantitates collagen and collagen-like protein samples with ease.

Novel Mutation in the COL11A1 Gene Causing Marshall-Stickler Syndrome in Three Generations of a Bulgarian Family.

Here we report the first familial case spread through at least three generations, genetically verified cases of Marshall-Stickler syndrome in Bulgaria. The proband, a 2-year-old girl, has craniofacial dysplasia, ocular hypertelorism, small saddle nose with a flat bridge and midface hypoplasia. The pedigree of the proband’s family showed that her father has the same clinical manifestations of the disease. In addition, her father presented with a tall, thin stature and mild hearing loss, manifested with aging. The same dysmorphological symptoms were presented by the paternal grandfather. Both patients, the 2-year-old girl and her father, have been diagnosed to carry Marshall-Stickler syndrome. The COL2A1 gene tested negative in the family. Based on the higher percentage of mutations in the COL2A1 gene, we analyzed this gene as the first target in the family. The COL2A1 gene tested negative, and we sequenced the gene further. A novel splice site mutation c.3474+1G>A was found in intron 44. This variant is related to the clinical presentation in the patient and her father. The c.3474+1G>A mutation results in altered splicing affects at the donor splice site of intron 44, which most probably gives a nonfunctional protein. The variant affects the major triple-helical domain that represents a mutation hot-spot for the gene.

Design Strategies to Tune the Structural and Mechanical Properties of Synthetic Collagen Hydrogels.

As an important component of biomaterials, collagen provides three-dimensional scaffolds and biological cues for cell adhesion and proliferation in tissue engineering. Recombinant collagen-like proteins, which were initially discovered in Streptococcus pyogenes and produced in heterologous hosts, have been chemically and genetically engineered for biomaterial applications. However, existing collagen-like proteins do not form gels, limiting their utility as biomaterials. Here, we present a series of rationally designed collagen-like proteins composed of a trimerization domain, triple-helical domains with various lengths, and a pair of heterotrimeric coiled-coil sequences attached to the N- and C-termini as adhesive ends. These designed proteins fold into triple helices and form self-supporting gels. As the triple-helical domains are lengthened, the gels become less stiff, pore sizes increase, and structural anisotropy decreases. Moreover, cell-culture assay confirms that the designed proteins are noncytotoxic. This study provides a design strategy for collagen-based biomaterials. The sequence variations reveal a relationship between the protein primary structure and material properties, where variations in the cross-linking density and association energies define the gelation of the protein network.

Causative variant profile of collagen VI-related dystrophy in Japan

BackgroundCollagen VI-related dystrophy spans a clinical continuum from severe Ullrich congenital muscular dystrophy to milder Bethlem myopathy. This disease is caused by causative variants in COL6A1, COL6A2, or COL6A3. Most reported causative variants are de novo; therefore, to identify possible associated causative variants, comprehensive large cohort studies are required for different ethnicities.MethodsWe retrospectively reviewed clinical information, muscle histology, and genetic analyses from 147 Japanese patients representing 130 families, whose samples were sent for diagnosis to the National Center of Neurology and Psychiatry between July 1979 and January 2020. Genetic analyses were conducted by gene-based resequencing, targeted panel resequencing, and whole exome sequencing, in combination with cDNA analysis.ResultsOf a total of 130 families with 1–5 members with collagen VI-related dystrophy, 120 had mono-allelic and 10 had bi-allelic variants in COL6A1, COL6A2, or COL6A3. Among them, 60 variants were in COL6A1, 57 in COL6A2, and 23 in COL6A3, including 37 novel variants. Mono-allelic variants were classified into four groups: missense (69, 58%), splicing (40, 33%), small in-frame deletion (7, 6%), and large genomic deletion (4, 3%). Variants in the triple helical domains accounted for 88% (105/120) of all mono-allelic variants.ConclusionsWe report the causative variant profile of a large set of Japanese cases of collagen VI-related dystrophy. This dataset can be used as a reference to support genetic diagnosis and variant-specific treatment.

Lysyl Oxidase‐like 2 Targets Specific Lysine residues within the 7S dodecamer of Collagen IV

Lysyl oxidase like 2 (LOXL2) has emerged as an attractive target for the development of therapies for different forms of tissue fibrosis including liver, lung, kidney and cancer-associated fibrosis. LOXL2 is a copper dependent enzyme that catalyzes the oxidative deamination of lysine residues to form lysyl aldehydes (allysine) in collagens. Allysines spontaneously condense into lysyl-derived crosslinks which are important for the deposition of collagens in the extracellular matrix (ECM). In our previous studies, we have established that LOXL2 promotes the formation of lysine-derived crosslinks in the 7S dodecamer, a triple-helical crosslinking domain formed by the overlap of four collagen IV molecules. Although the specific location lysine residues targeted by lysyl oxidase have been described for fibrillar collagens, the equivalent information for 7S dodecamer of collagen IV is unknown. To better understand the mechanism by which LOXL2 mediates the crosslinking of collagen IV, we developed a fluorometric assay to detect amine oxidase activity using 7S dodecamer as substrate. We characterized the ability of LOXL2 to deaminate lysine residues in two forms of 7S substrate: the wild-type crosslinked 7S substrate and an uncrosslinked form purified from ECM derived from cells lacking LOXL2. Surprisingly, the enzyme showed little to no activity with crosslinked 7S dodecamer whereas a pronounced enzyme activity was observed with the uncrosslinked form of the 7S substrate. As trypsin digestion indicated that not all lysine residues present in the wild type 7S dodecamer take part in crosslinking, we hypothesized that the LOXL2 targets specific lysine residues. To identify specific lysine residues, uncrosslinked 7S dodecamers were oxidized with LOXL2 and newly formed allysines were labeled with a biotin tag. Optimization of biotinylation reaction was monitored by Streptavidin-HRP followed by enhanced chemiluminescence (ECL). After trypsin digestion of 7S dodecamers, biotinylated peptides containing allysine residues were identified by mass spectrometry. We discuss the potential rol of the identified allysines in the alignment of the four triple-helical molecules forming the 7S dodecamer. A better understanding of the mechanism by which LOXL2 promotes collagen IV crosslinking will provide the foundations for the development of effective therapies against tissue fibrosis.

Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta

Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI.

Balancing Cost and Accuracy in Quantum Mechanical Simulations on Collagen Protein Models.

Collagen proteins are spread in almost every vertebrate's tissue with mechanical function. The defining feature of this fundamental family of proteins is its well-known collagen triple-helical domain. This helical domain can have different geometries, varying in helical elongation and interstrands contact, as a function of the amino acidic composition. The helical geometrical features play an important role in the interaction of the collagen protein with cell receptors, but for the vast majority of collagen compositions, these geometrical features are unknown. Quantum mechanical (QM) simulations based on the density functional theory (DFT) provide a robust approach to characterize the scenario on the collagen composition-structure relationships. In this work, we analyze the role of the adopted computational method in predicting the collagen structure for two purposes. First, we look for a cost-effective computational approach to apply to a large-scale composition-structure analysis. Second, we attempt to assess the robustness of the predictions by varying the QM methods. Therefore, we have run geometry optimization on periodic models of the collagen protein using a variety of approaches based on the most commonly used DFT functionals (PBE, HSE06, and B3LYP) with and without dispersion correction (D3ABC). We have coupled these methods with several different basis sets, looking for the highest accuracy/cost ratio. Furthermore, we have studied the performance of the composite HF-3c method and the semiempirical GFN1-xTB method. Our results identify a computational recipe that is potentially capable of predicting collagen structural features in line with DFT simulations, with orders of magnitude reduced computational cost, encouraging further investigations on the topic.

Collagen VI Muscle Disorders: Mutation Types, Pathogenic Mechanisms and Approaches to Therapy.

Mutations in the genes encoding the major collagen VI isoform, COL6A1, COL6A2 and COL6A3, are responsible for the muscle disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. These disorders form a disease spectrum from mild to severe. Dominant and recessive mutations are found along the entire spectrum and the clinical phenotype is strongly influenced by the way mutations impede collagen VI protein assembly. Most mutations are in the triple helical domain, towards the N-terminus and they compromise microfibril assembly. Some mutations are found outside the helix in the C- and N-terminal globular domains, but because these regions are highly polymorphic it is difficult to discriminate mutations from rare benign changes without detailed structural and functional studies. Collagen VI deficiency leads to mitochondrial dysfunction, deficient autophagy and increased apoptosis. Therapies that target these consequences have been tested in mouse models and some have shown modest efficacy in small human trials. Antisense therapies for a common mutation that introduces a pseudoexon show promise in cell culture but haven't yet been tested in an animal model. Future therapeutic approaches await new research into how collagen VI deficiency signals downstream consequences.