Porin Proteins in Mitochondria from Rat Pancreatic Islet Cells and White Adipocytes

The binding of hexo-/glucokinase and glycerol kinase to mitochondria via the channel forming protein, porin, in pancreatic islet β-cells and adipocytes, was recently proposed to participate in nutritional signaling, glucose sensing, and the control of high-energy phosphate distribution and oxidative phosphorylation. In this study we demonstrate that polyclonal antisera against purified rat liver porin recognize unique proteins in rat pancreatic islets, adipocytes, and RINm5F cells, each with an apparent Mr about 2000 smaller than that of liver porin. Immunoblotting of subcellular fractions, the purity of which has been controlled by the distribution of marker proteins, revealed the mitochondrial localization of the cross-reacting proteins. Their enrichment with a method used for the purification of porin proteins, the characteristic behavior during isoelectric focusing, and the specific binding of rat liver hexokinase and glycerol kinase to phospholipid vesicles containing the purified cross-reacting β-cell or adipocyte proteins strongly suggest their identity with mitochondrial porin. The subtle differences in the apparent Mr and charge heterogeneity raise the possibility of the existence of porin isoforms expressed in a tissue-specific manner. Anti-porin antisera coimmunoprecipitated hexo-/glucokinase from rat insulinoma cell (RINm5F) and adipocyte mitochondria as determined by subsequent immunoblotting of the immunoprecipitates with polyclonal antisera against yeast hexokinase and rat liver glucokinase, respectively. This indicates that some rat pancreatic glucokinase (54 kDa) and liver hexokinase (102 kDa), respectively, is bound to mitochondrial porin. The major portion of the bound fraction is released from mitochondria after treatment with glucose 6-phosphate. Incubation of RINm5F and fat cells with the insulin releasing sulfonylurea drug, glimepiride (20 nM and 5 μM, respectively) for 30 min reduces the amount of hexo-/glucokinase associated with mitochondria and porin to about 50-30%. The reduced kinase binding activity of porin is preserved after isolation of porin from glimepiride-treated cells, reconstitution into phospholipid vesicles and assaying for glucose 6-phosphate inhibitable binding of rat liver hexokinase. The sulfonylurea tolbutamide (20 μM and 5 mM) is significantly less effective. The sulfonylurea-induced inhibition of hexo-/glucokinase binding to mitochondrial porin does not require glucose metabolism or Ca2+ influx into the cells. These data suggest that the sulfonylurea glimepiride, which is thought to inhibit the ATP-regulated K+-channel in β-cells, may have, in addition, an intracellular site of action in pancreatic islet and adipocyte cells at the level of regulation of gluco-/hexokinase binding to mitochondrial porin

Assessment of Bones Deficient in Fibrillin-1 Microfibrils Reveals Pronounced Sex Differences.

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFβ) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFβ signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches

Early-Onset Osteoporosis : Rare Monogenic Forms Elucidate the Complexity of Disease Pathogenesis Beyond Type I Collagen

Early-onset osteoporosis (EOOP), characterized by low bone mineral density (BMD) and fractures, affects children, premenopausal women and men agedPeer reviewe

Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis

VarFish: comprehensive DNA variant analysis for diagnostics and research

VarFish is a user-friendly web application for the quality control, filtering, prioritization, analysis, and user-based annotation of DNA variant data with a focus on rare disease genetics. It is capable of processing variant call files with single or multiple samples. The variants are automatically annotated with population frequencies, molecular impact, and presence in databases such as ClinVar. Further, it provides support for pathogenicity scores including CADD, MutationTaster, and phenotypic similarity scores. Users can filter variants based on these annotations and presumed inheritance pattern and sort the results by these scores. Variants passing the filter are listed with their annotations and many useful link-outs to genome browsers, other gene/variant data portals, and external tools for variant assessment. VarFish allows users to create their own annotations including support for variant assessment following ACMG-AMP guidelines. In close collaboration with medical practitioners, VarFish was designed for variant analysis and prioritization in diagnostic and research settings as described in the software's extensive manual. The user interface has been optimized for supporting these protocols. Users can install VarFish on their own in-house servers where it provides additional lab notebook features for collaborative analysis and allows re-analysis of cases, e.g. after update of genotype or phenotype databases

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino aciddependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.Peer reviewe

Zebrafish as a model to investigate a biallelic gain-of-function variant in MSGN1, associated with a novel skeletal dysplasia syndrome

BACKGROUND/OBJECTIVES: Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos. RESULTS: The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix-Loop-Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function. CONCLUSION: In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors

Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7‐related osteopetrosis

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl-/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.Peer reviewe

Impaired proteoglycan glycosylation, elevated TGF-β signaling, and abnormal osteoblast differentiation as the basis for bone fragility in a mouse model for gerodermia osteodysplastica

<div><p>Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. <i>GORAB</i>, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the <i>Gorab</i>^<i>Null</i> full knockout, <i>Gorab</i> was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only <i>Gorab</i>^Prx1 and <i>Gorab</i>^Runx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of <i>Gorab</i>^<i>Null</i> mutants and in bone of <i>Gorab</i>^Prx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from <i>Gorab</i>^<i>Null</i> mutants. In bone from <i>Gorab</i>^Prx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured <i>GORAB</i>-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in <i>Gorab</i>^Prx1 bone tissue leading to enhanced downstream signalling, which was reproduced in <i>GORAB</i>-deficient fibroblasts. Our data suggest that the loss of <i>Gorab</i> primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.</p></div