Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

IPIP27A cooperates with OCRL to support endocytic traffic in the zebrafish pronephric tubule.

Endocytosis is a fundamentally important process through which material is internalized into cells from the extracellular environment. In the renal proximal tubule, endocytosis of the abundant scavenger receptor megalin and its co-receptor cubilin play a vital role in retrieving low molecular weight proteins from the renal filtrate. Although we know much about megalin and its ligands, the machinery and mechanisms by which the receptor is trafficked through the endosomal system remain poorly defined. In this study, we show that inositol phosphatase interacting protein of 27 kDa (Ipip27A), an interacting partner of the Lowe syndrome protein oculocerebrorenal syndrome of Lowe (OCRL), is required for endocytic traffic of megalin within the proximal renal tubule of zebrafish larvae. Knockout of Ipip27A phenocopies the endocytic phenotype seen upon loss of OCRL, with a deficit in uptake of both fluid-phase and protein cargo, which is accompanied by a reduction in megalin abundance and altered endosome morphology. Rescue and co-depletion experiments indicate that Ipip27A functions together with OCRL to support proximal tubule endocytosis. The results therefore identify Ipip27A as a new player in endocytic traffic in the proximal tubule in vivo and support the view that defective endocytosis underlies the renal tubulopathy in Lowe syndrome and Dent-2 disease.

A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Heterologous Expression and Characterization of a Novel Mesophilic Maltogenic α-Amylase AmyFlA from Flavobacterium sp. NAU1659.

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli. Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50 °C in 50 mM phosphate buffer (pH 6.0). The Km and Vmax values of AmyFlA were respectively 3.15 mg/ml and 566.36 µmol·ml-1·min-1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca2+, indicating that AmyFlA was a Ca2+-dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup.

Temperature-dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9-Tim10 complex.

Protein-protein interaction is a fundamental process in all major biological processes. The hexameric Tim9-Tim10 (translocase of inner membrane) complex of the mitochondrial intermembrane space plays an essential chaperone-like role during import of mitochondrial membrane proteins. However, little is known about the functional mechanism of the complex because the interaction is weak and transient. This study investigates how electrostatic and hydrophobic interactions affect the conformation and function of the complex at physiological temperatures, using both experimental and computational methods. The results suggest that, first, different complex conformational states exist at equilibrium, and the major difference between these states is the degree of hydrophobic interactions. Second, the conformational change mimics the biological activity of the complex as measured by substrate binding at the same temperatures. Finally, molecular dynamics simulation and detailed energy decomposition analysis provided supporting evidence at the atomic level for the presence of an excited state of the complex, the formation of which is largely driven by the disruption of hydrophobic interactions. Taken together, this study indicates that the dynamics of the hydrophobic residues plays an important role in regulating the function of the Tim9-Tim10 complex.

Association of CYP11B2 gene polymorphism with ischemic stroke in the north Chinese Han population.

BACKGROUND: Genetic variations of renin-angiotensin-aldosterone system play an important role in the pathogenesis of hypertension and stroke. AIM: To investigate the -344C/T and intron 2 conversion polymorphisms of aldosterone synthase gene (CYP11B2) for an association with stroke and hypertension in the North Chinese Han population. MATERIALS AND METHODS: This case-control study included 332 patients and 250 controls. Genotypes of -344C/T polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the intron 2 conversion polymorphism was genotyped using two separated PCRs. RESULTS: There were significant differences in genotype frequencies of -344C/T polymorphism between stroke patients and controls (P = 0.002). An association was found between TT genotype and ischemic stroke [odds ratio = 1.572, 95%CI (1.095-2.258), P = 0.014]. However, there was no significant association of intron 2 polymorphism with stroke. Furthermore, when the ischemic stroke patients were classified according to Trial of Org 10172 in Acute Stroke Treatment classification, TT genotype was found to be associated with large artery atherosclerosis [odds ratio = 1.747, 95%CI (1.182-2.584), P = 0.005] and small vessel disease [odds ratio = 1.781, 95%CI (1.134-2.796), P = 0.012]. The intron 2 polymorphism failed to show relationship with any specific stroke subtype. CONCLUSIONS: Our findings suggest a significant association of CYP11B2 (-344C/T) polymorphism with stroke but intron 2 polymorphism is not associated with increased stroke susceptibility.

Pacsin2 is required for endocytosis in the zebrafish pronephric tubule.

Endocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the scavenger receptor megalin and its co-receptor cubilin mediate endocytosis of low molecular weight proteins from the renal filtrate. However, the extent to which megalin endocytosis relies on different components of the trafficking machinery remains relatively poorly defined in vivo. In this study, we identify a functional requirement for the F-BAR protein pacsin2 in endocytosis in the renal proximal tubule of zebrafish larvae. Pacsin2 is expressed throughout development and in all zebrafish tissues, similar to the mammalian orthologue. Within renal tubular epithelial cells, pacsin2 is enriched at the apical pole where it is localised to endocytic structures. Loss of pacsin2 results in reduced endocytosis within the proximal tubule, which is accompanied by a reduction in the abundance of megalin and endocytic organelles. Our results indicate that pacsin2 is required for efficient endocytosis in the proximal tubule, where it likely cooperates with other trafficking machinery to maintain endocytic uptake and recycling of megalin.

IPIP27 Coordinates PtdIns(4,5)P2 Homeostasis for Successful Cytokinesis.

During cytokinesis, an actomyosin contractile ring drives the separation of the two daughter cells. A key molecule in this process is the inositol lipid PtdIns(4,5)P2, which recruits numerous factors to the equatorial region for contractile ring assembly. Despite the importance of PtdIns(4,5)P2 in cytokinesis, the regulation of this lipid in cell division remains poorly understood. Here, we identify a role for IPIP27 in mediating cellular PtdIns(4,5)P2 homeostasis. IPIP27 scaffolds the inositol phosphatase oculocerebrorenal syndrome of Lowe (OCRL) by coupling it to endocytic BAR domain proteins. Loss of IPIP27 causes accumulation of PtdIns(4,5)P2 on aberrant endomembrane vacuoles, mislocalization of the cytokinetic machinery, and extensive cortical membrane blebbing. This phenotype is observed in Drosophila and human cells and can result in cytokinesis failure. We have therefore identified IPIP27 as a key modulator of cellular PtdIns(4,5)P2 homeostasis required for normal cytokinesis. The results indicate that scaffolding of inositol phosphatase activity is critical for maintaining PtdIns(4,5)P2 homeostasis and highlight a critical role for this process in cell division.

Mesenchymal stem cell transplantation as an effective treatment strategy for ischemic stroke in Asia: a meta-analysis of controlled trials.

OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of the mesenchymal stem cell (MSC) therapy in patients with ischemic stroke (IS). MATERIALS AND METHODS: Clinical trials involved in this research were searched from PubMed, Web of Science, Cochrane Library, Embase, Wanfang and CNKI database. Therapeutic effects of MSC therapy were assessed according to National Institutes of Health Stroke Scale (NIHSS), Barthel index (BI), Fugl-Meyer Assessment (FMA) and Functional Independence Measure (FIM), and its safety was evaluated based on adverse events. RESULTS: This research covered 23 trials including 1,279 IS patients. Based on our analysis, the overall condition of IS patients significantly improved after MSC therapy, indicated by decreased NIHSS and increased BI, FMA and FIM scores. Our analysis also showed that the treatment effects in the MSC transplantation group were superior to those in the control group (routine medication therapy) with statistical significance for NIHSS (1 month after therapy: odds ratio [OR]=-1.92, CI=-3.49 to -0.34, P=0.02; 3 months after therapy: OR=-2.65, CI=-3.40 to -1.90, P<0.00001), BI (1 month after therapy: OR=0.99, CI=0.19-1.79, P=0.02; 6 months after therapy: OR=10.10, CI=3.07-17.14, P=0.005), FMA (3 months after therapy: OR=10.20, CI=3.70-16.70, P=0.002; 6 months after therapy: OR=10.82, CI=6.45-15.18, P<0.00001) and FIM (1 month after therapy: OR=15.61, CI=-0.02 to 31.24, P=0.05; 6 months after therapy: OR=16.56, CI=9.06-24.06, P<0.0001). No serious adverse events were reported during MSC therapy. CONCLUSION: MSC therapy is safe and effective in treating IS by improving the neurological deficits, motor function and daily life quality of patients.

OCRL1 engages with the F-BAR protein pacsin 2 to promote biogenesis of membrane-trafficking intermediates.

Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)-containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2-dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes.

Role of reverse phenotyping in interpretation of next generation sequencing data and a review of INPP5E related disorders.

INTRODUCTION: Next Generation Sequencing (NGS) is a useful tool in diagnosis of rare disorders but the interpretation of data can be challenging in clinical settings. We present results of extended studies on a family of multiple members with global developmental delay and learning disability, where another research group postulated the underlying cause to be a homozygous RABL6 missense variant. METHODS AND RESULTS: Using data from the Exome Variant Server, we show that missense RABL6 variants are unlikely to cause early onset rare developmental disorder. Protein structural analysis, cellular functional studies and reverse phenotyping proved that the condition in this family is due to a homozygous INPP5E mutation. An in-depth review of mutational and phenotypic spectrum associated with INPP5E demonstrated that mutations in this gene lead to a range of cilliopathy-phenotypes. DISCUSSION: We use this study as an example to demonstrate the importance of careful clinical evaluation of multiple family members, reverse phenotyping, considering the unknown phenotypic variability of rare diseases, utilizing publically available genomic databases and conducting appropriate bioinformatics and functional studies while interpreting results from NGS in uncertain cases. We emphasize that interpretation of NGS data is an iterative process and its dynamic nature should be explained to patients and families. Our study shows that developmental delay, intellectual disability, hypotonia and ocular motor apraxia are common in INPP5E-related disorders and considerable intra-familial phenotypic variability is possible. We have compiled the INPP5E mutational spectrum and provided novel insights into their molecular mechanisms.

Zinc can play chaperone-like and inhibitor roles during import of mitochondrial small Tim proteins.

Zinc is an essential cofactor required for the function of approximately 8% of the yeast and 10% of the human proteome. All of the "small Tim" proteins of the mitochondrial intermembrane space contain a strictly conserved "twin CX(3)C" zinc finger motif, which can bind zinc ions in the Cys-reduced form. We have shown previously that although disulfide bond formation is essential for the function of these proteins in mitochondria, only reduced proteins can be imported into mitochondria (Lu, H., Allen, S., Wardleworth, L., Savory, P., and Tokatlidis, K. (2004) J. Biol. Chem. 279, 18952-18958 and Morgan, B., and Lu, H. (2008) Biochem. J. 411, 115-122). However, the role of zinc during the import of these proteins is unclear. This study shows that the function of zinc is complex. It can play a thiol stabilizer role preventing oxidative folding of the small Tim proteins and maintaining the proteins in an import-competent form. On the other hand, zinc-bound forms cannot be imported into mitochondria efficiently. Furthermore, our results show that zinc is a powerful inhibitor of Erv1, an essential component of the import pathway used by the small Tim proteins. We propose that zinc plays a chaperone-like role in the cytosol during biogenesis of the small Tim proteins and that the proteins are imported into mitochondria through the apo-forms.