Mechanistic behaviour and molecular interactions of heat shock protein 47 (HSP47)

This project involves the study of heat shock protein 47 (HSP47), which is a molecular chaperone crucial for collagen biosynthesis. It exhibits a high degree of sequence homology with members of the serine protease inhibitor (serpin) superfamily, though HSP47 does not possess the inhibitory activity. It is a single-substrate chaperone, and binds only to collagen. ‘Knock-out’ of the hsp47 gene impairs the secretion of correctly folded collagen triple helix molecules leading to embryonic lethality in mice. Thus the aim of this project was to elucidate the specific mechanism that governs the binding to and release from collagen at the molecular level, known as the ‘pH-switch mechanism’. Emphasis is given on histidine (His) residues as the HSP47-collagen dissociation pH is similar to the pKa of the imidazole side chain of His residues. Site directed mutagenesis was used to mutate surface His residues, based on a mouse HSP47 homology model. The effects of the mutations on the behaviour of HSP47 were then assessed by collagen binding assays and structural analyses with circular dichroism (CD). All mutants were found to have good solubility and retain their binding ability to collagen like wild-type HSP47 in batch assay, but perturbed behaviour was seen in column experiment. Mutation of His residue at position 191 (H191) causes the shift in the collagen dissociation pH, while mutation of H197 and/or 198 disrupt the specific HSP47-collagen interaction. H191, 197 and 198 are predicted to be located in the region near the C-terminus of strand 3 of β-sheet A (s3A) in the homology model, a region specifically known as the ‘breach cluster’ in serpin nomenclature. The extent of conformational rearrangement of this region was further investigated by means of intrinsic tryptophan fluorescence spectroscopy using a series of single tryptophan (Trp) mutants. Results from analyses performed on the mutants did not contradict the observation seen in His mutational work, as Trp residues in the ‘breach’ cluster are likely to be located in the dynamic region of HSP47 pH-triggered conformational change. In conclusion, this study establishes the importance of His residues in the ‘breach cluster’ to HSP47 pH-switch behaviour. Finally, a model for HSP47 pH-switch mechanism was proposed from data obtained via mutagenesis experiments. The model is hoped to assist future research into HSP47 cellular behaviour and will also be of great use in therapeutic applications involving the molecular chaperone

Helical mutations in type I collagen that affect the processing of the amino-propeptide result in an Osteogenesis Imperfecta/Ehlers-Danlos Syndrome overlap syndrome

Background: Whereas mutations affecting the helical domain of type I procollagen classically cause Osteogenesis Imperfecta (OI), helical mutations near the amino (N)-proteinase cleavage site have been suggested to result in a mixed OI/Ehlers-Danlos syndrome (EDS)-phenotype. Methods: We performed biochemical and molecular analysis of type I (pro-) collagen in a cohort of seven patients referred with a clinical diagnosis of EDS and showing only subtle signs of OI. Transmission electron microscopy of the dermis was available for one patient. Results: All of these patients harboured a COL1A1 / COL1A2 mutation residing within the most N-terminal part of the type I collagen helix. These mutations affect the rate of type I collagen N-propeptide cleavage and disturb normal collagen fibrillogenesis. Importantly, patients with this type of mutation do not show a typical OI phenotype but mainly present as EDS patients displaying severe joint hyperlaxity, soft and hyperextensible skin, abnormal wound healing, easy bruising, and sometimes signs of arterial fragility. In addition, they show subtle signs of OI including blue sclerae, relatively short stature and osteopenia or fractures. Conclusion: Recognition of this distinct phenotype is important for accurate genetic counselling, clinical management and surveillance, particularly in relation to the potential risk for vascular rupture associated with these mutations. Because these patients present clinical overlap with other EDS subtypes, biochemical collagen analysis is necessary to establish the correct diagnosis

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing read-through and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived from patients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1

Genotype-phenotype correlation study in 364 osteogenesis imperfecta Italian patients

Osteogenesis imperfecta (OI) is a rare genetic disorder of the connective tissue and 90% of cases are due to dominant mutations in COL1A1 and COL1A2 genes. To increase OI disease knowledge and contribute to patient follow-up management, a homogeneous Italian cohort of 364 subjects affected by OI types I-IV was evaluated. The study population was composed of 262 OI type I, 24 type II, 39 type III, and 39 type IV patients. Three hundred and nine subjects had a type I collagen affecting function mutations (230 in α1(I) and 79 in α2(I)); no disease-causing changes were noticed in 55 patients. Compared with previous genotype-phenotype OI correlation studies, additional observations arose: a new effect for α1- and α2-serine substitutions has been pointed out and heart defects, never considered before, resulted associated to quantitative mutations (P = 0.043). Moreover, some different findings emerged if compared with previous literature; especially, focusing the attention on the lethal form, no association with specific collagen regions was found and most of variants localized in the previously reported "lethal clusters" were causative of OI types I-IV. Some discrepancies have been highlighted also considering the "50-55 nucleotides rule," as well as the relationship between specific collagen I mutated region and the presence of dentinogenesis imperfecta and/or blue sclera. Despite difficulties still present in defining clear rules to predict the clinical outcome in OI patients, this study provides new pieces for completing the puzzle, also thanks to the inclusion of clinical signs never considered before and to the large number of OI Italian patients

The kunitz domain protein BLI-5 plays a functionally conserved role in cuticle formation in a diverse range of nematodes

The cuticle of parasitic nematodes performs many critical functions and is essential for proper development and for protection from the host immune response. The biosynthesis, assembly, modification and turnover of this exoskeleton have been most extensively studied in the free-living nematode, Caenorhabditis elegans, where it represents a complex multi-step process involving a whole suite of enzymes. The biosynthesis of the cuticle has an additional level of complexity, as many of the enzymes also require additional proteins to aid their activation and selective inhibition. Blister-5 (BLI-5) represents a protein with a kunitz-type serine protease interacting domain and is involved in cuticle collagen biosynthesis in C. elegans, through its interaction with subtilisin-like processing enzymes (such as BLI-4). Mutation of the bli-5 gene causes blistering of the collagenous adult cuticle. Homologues of BLI-5 have been identified in several parasitic species that span different nematode clades. In this study, we molecularly and biochemically characterize BLI-5 homologues from the clade V nematodes C. elegans and Haemonchus contortus and from the clade III filarial nematode Brugia malayi. The nematode BLI-5 orthologues possess a shared domain structure and perform similar in vitro and in vivo functions, performing important proteolytic enzyme functions. The results demonstrate that the bli-5 genes from these diverse parasitic nematodes are able to complement a C. elegansbli-5 mutant and thereby support the use of the C. elegans model system to examine gene function in the experimentally less-amenable parasitic species

Hearing impairment in Stickler syndrome: a systematic review

BACKGROUND: Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely COL2A1, COL11A1 and COL11A2 (autosomal dominant inheritance), and COL9A1 and COL9A2 (autosomal recessive inheritance). The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype. METHODS: English-language literature was reviewed through searches of PubMed and Web of Science, in order to find relevant articles describing auditory features in Stickler patients, along with genotype. Prevalences of hearing loss are calculated and correlated with the different affected genes and type of mutation. RESULTS: 313 patients (102 families) individually described in 46 articles were included. Hearing loss was found in 62.9%, mostly mild to moderate when reported. Hearing impairment was predominantly sensorineural (67.8%). Conductive (14.1%) and mixed (18.1%) hearing loss was primarily found in young patients or patients with a palatal defect. Overall, mutations in COL11A1 (82.5%) and COL11A2 (94.1%) seem to be more frequently associated with hearing impairment than mutations in COL2A1 (52.2%). CONCLUSIONS: Hearing impairment in patients with Stickler syndrome is common. Sensorineural hearing loss predominates, but also conductive hearing loss, especially in children and patients with a palatal defect, may occur. The distinct disease-causing collagen genes are associated with a different prevalence of hearing impairment, but still large phenotypic variation exists. Regular auditory follow-up is strongly advised, particularly because many Stickler patients are visually impaired

Ancient fossil specimens of extinct species are genetically more distant to an outgroup than extant sister species are

There exists a remarkable correlation between genetic distance and time of species divergence as inferred from fossil records. This observation has provoked the molecular clock hypothesis. However, data inconsistent with the hypothesis have steadily accumulated in recent years from studies on extant organisms. Here the published DNA and protein sequences from ancient fossil specimens were examined to see if they would support the molecular clock hypothesis. The hypothesis predicts that ancient specimens cannot be genetically more distant to an outgroup than extant sister species are. Also, two distinct ancient specimens cannot be genetically more distant than their extant sister species are. The findings here did not support these predictions. Neanderthals are more distant to chimpanzees and gorillas than modern humans are. Dinosaurs are more distant to frogs than extant birds are. Mastodons are more distant to opossums than other placental mammals are. The genetic distance between dinosaurs and mastodons is greater than that between extant birds and mammals. Therefore, while the molecular clock hypothesis is consistent with some data from extant organisms, it has yet to find support from ancient fossils

Genetic Suppression of Basement Membrane Defects in Caenorhabditis elegans by Gain of Function in Extracellular Matrix and Cell-Matrix Attachment Genes.

Basement membranes are extracellular matrices essential for embryonic development in animals. Peroxidasins are extracellular peroxidases implicated in the unique sulfilimine cross-links between type IV basement membrane collagens. Loss of function in the Caenorhabditis elegans peroxidasin PXN-2 results in fully penetrant embryonic or larval lethality. Using genetic suppressor screening, we find that the requirement for PXN-2 in development can be bypassed by gain of function in multiple genes encoding other basement membrane components, or proteins implicated in cell-matrix attachment. We identify multiple alleles of let-805, encoding the transmembrane protein myotactin, which suppress phenotypes of pxn-2 null mutants and of other basement membrane mutants such as F-spondin/spon-1 These let-805 suppressor alleles cause missense alterations in two pairs of FNIII repeats in the extracellular domain; they act dominantly and have no detectable phenotypes alone, suggesting they cause gain of function. We also identify suppressor missense mutations affecting basement membrane components type IV collagen (emb-9, let-2) and perlecan (unc-52), as well as a mutation affecting spectraplakin (vab-10), a component of the epidermal cytoskeleton. These suppressor alleles do not bypass the developmental requirement for core structural proteins of the basement membrane such as laminin or type IV collagen. In conclusion, putative gain-of-function alterations in matrix proteins or in cell-matrix receptors can overcome the requirement for certain basement membrane proteins in embryonic development, revealing previously unknown plasticity in the genetic requirements for the extracellular matrix

Structure-mechanics relationships of collagen fibrils in the Osteogenesis Imperfecta Mouse model

The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry.United States. Dept. of Defense. Presidential Early Career Award for Scientists and EngineersNational Science Foundation (U.S.) (CAREER Award

Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are α2β2 tetramers, in which the β subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the <i>Caenorhabditis</i> <i>elegans</i> catalytic α subunit isoforms PHY-1 and PHY-2 and the β subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)<sub>2</sub> tetramer is the major form, while PHY-1/PDI- 2 and PHY-2/PDI-2 dimers are also assembled but less efficiently. Cloning and characterization of the orthologous subunits from the closely related nematode <i>Caenorhabditis</i> <i>briggsae</i> revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the <i>C. briggsae</i> and <i>C. elegans</i> subunits. In addition to a PHY-1/PHY-2(PDI-2)<sub>2</sub> tetramer and a PHY-1/PDI-2 dimer, an active (PHY- 2)<sub>2</sub>(PDI-2)<sub>2</sub> tetramer was formed in <i>C. briggsae</i> instead of a PHY-2/PDI-2 dimer. Site-directed mutagenesis studies and generation of inter-species hybrid polypeptides showed that the N-terminal halves of the <i>Caenorhabditis</i> PHY-2 polypeptides determine their assembly properties. Genetic disruption of <i>C. briggsae phy-1</i> (<i>Cb-dpy-18</i>) via a <i>Mos1</i> insertion resulted a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding <i>C. elegans</i> mutants (<i>Ce-dpy-18</i>). <i>C. briggsae</i> <i>phy-2</i> RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in <i>phy-1</i> mutants. Genetic complementation of the <i>C. briggsae</i> and <i>C. elegans</i> <i>phy-1</i> mutants was achieved by injection of a wild type <i>phy-1</i> gene from either species