MicroR-380-3p Reduces Sepsis-Induced Acute Kidney Injury via Regulating RAB1P to Restrain NF-κB Pathway.

Septic acute kidney injury (AKI) is a common complication in critically ill patients with high morbidity and mortality. This study intends to clarify the clinical value and molecular mechanism of microR-380-3p in septic AKI by recruiting patients with septic AKI and establishing septic AKI cell models. Patients with septic AKI were included and human kidney-2 (HK-2) cells were induced by lipopolysaccharide (LPS) to construct the AKI cell model of sepsis. The expression of microR-380-3p was detected by quantitative real-time RT-PCR (qRT-PCR). The expression of Bax, cleaved caspase 3, Bcl-2, p65, and p-p65 was detected by Western blot. The contents of inflammation and oxidation were determined by commercial kits. Bioinformatics predicted the binding target of microR-380-3p and a dual luciferase reporting system was used to verify the regulatory relationship between microR-380-3p and RAP1B. The concentration of microR-380-3p was elevated in patients with septic AKI and appeared to be a biomarker for these patients. Silenced microR-380-3p reversed the damage of LPS on HK-2 cells via promoting viability, inhibiting apoptosis, inflammation, and oxidation. RAP1B was a target of microR-380-3p and microR-380-3p exerted targeted inhibition of RAP1B expression level. Down-regulation of RAP1B reversed the influence of silenced microR-380-3p on HK-2 cells. MicroR-380-3p/RAP1B participated in activating the NF-κB pathway. MicroR-380-3p down-regulated RAP1B to exacerbate septic AKI, providing a potential therapeutic biomarker for septic AKI.

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (ER)-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Rab29 activation of the Parkinson's disease-associated LRRK2 kinase.

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

Circ_0026123 promotes cisplatin resistance and progression of ovarian cancer by upregulating RAB1A through sequestering miR-543.

BACKGROUND: Circular RNAs can act as critical regulators in the tumorigenesis and chemoresistance of ovarian cancer (OC). Herein, this work aimed to probe the function and mechanism of circ_0026123 in the cisplatin (DDP) resistance and progression of OC and its potential value in the clinic. METHODS: The quantitative real-time PCR and western blotting were used to detect the levels of RNAs and proteins. In vitro experiments were conducted using CCK-8, EdU, transwell, tube formation assays and flow cytometry. Mouse subcutaneous xenograft model was used for in vivo experiments. The interaction between circ_0026123 or RAB1A (Ras-related protein Rab-1A) and miR-543 was confirmed using dual-luciferase reporter and RNA immunoprecipitation assays. RESULTS: Circ_0026123 expression was higher in DDP-resistant OC tissues and cells. Silencing of circ_0026123 dramatically boosted the sensitivity of DDP-resistant OC cells to DDP, as well as inhibited cell growth, angiogenesis, invasion and migration abilities in vitro. Circ_0026123 functionally targeted miR-543, and knockdown of miR-543 reversed the impacts of circ_0026123 deficiency on DDP sensitivity and the malignant behaviors of DDP-resistant OC cells. RAB1A was a target of miR-543, RAB1A overexpression attenuated the inhibitory functions of miR-543 on DDP resistance and the malignant phenotypes of DDP-resistant OC cells. Preclinically, lentivirus-mediated circ_0026123 downregulation also suppressed OC growth and enhanced DDP cytotoxicity in vivo. CONCLUSION: Our study demonstrated that circ_0026123 acted as a sponge for miR-543 to elevate RAB1A expression, thus promoting cisplatin resistance and tumorigenesis in ovarian cancer.

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas.

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas.

Circ_0061140 Contributes to the Malignant Progression in Ovarian Cancer Cells by Mediating the RAB1A Level Through Sponging miR-361-5p.

Ovarian cancer (OC) progression is related to many functional molecules, including circular RNAs (circRNAs). Hsa_circ_0061140 (circ_0061140) promoted cell growth and metastasis in OC. The aim of this study was to explore a specific functional mechanism of circ_0061140. Reverse transcription-quantitative polymerase chain reaction was performed for expression analysis of circ_0061140, microRNA-361-5p (miR-361-5p), and Ras-like protein in rat brain 1A (RAB1A). Cell proliferation was determined using Cell Counting Kit-8 assay, EdU assay, and colony formation assay. The migration and invasion were assessed through transwell assay. Tube formation assay was used for angiogenesis analysis. Cell apoptosis was evaluated using flow cytometry. The protein levels of epithelial-to-mesenchymal transition (EMT) markers and RAB1A were detected via western blot. Target analysis was performed by dual-luciferase reporter assay and RNA immunoprecipitation assay. In vivo research was conducted using xenograft model. The circ_0061140 level was upregulated in OC samples and cells. Downregulation of circ_0061140 impeded proliferation, migration, invasion, EMT, and angiogenesis of OC cells. Circ_0061140 directly interacted with miR-361-5p to act as a miRNA sponge. The miR-361-5p inhibition reversed the si-circ_0061140-induced anti-tumor function in OC cells. RAB1A was a downstream target of miR-361-5p, and miR-361-5p served as a tumor repressor in OC via inhibiting the level of RAB1A. Circ_0061140 could increase the RAB1A expression by sponging miR-361-5p in OC cells. Circ_0061140 also facilitated tumorigenesis in vivo through targeting the miR-361-5p/RAB1A axis. All results demonstrated that circ_0061140 promoted OC development by inhibiting miR-361-5p to upregulate the expression of RAB1A.

The Small GTPase FgRab1 Plays Indispensable Roles in the Vegetative Growth, Vesicle Fusion, Autophagy and Pathogenicity of Fusarium graminearum.

Rab GTPases are key regulators of membrane and intracellular vesicle transports. However, the biological functions of FgRab1 are still unclear in the devastating wheat pathogen Fusarium graminearum. In this study, we generated constitutively active (CA) and dominant-negative (DN) forms of FgRAB1 from the wild-type PH-1 background for functional analyses. Phenotypic analyses of these mutants showed that FgRab1 is important for vegetative growth, cell wall integrity and hyphal branching. Compared to the PH-1 strain, the number of spores produced by the Fgrab1DN strain was significantly reduced, with obviously abnormal conidial morphology. The number of septa in the conidia of the Fgrab1DN mutant was fewer than that observed in the PH-1 conidia. Fgrab1DN was dramatically reduced in its ability to cause Fusarium head blight symptoms on wheat heads. GFP-FgRab1 was observed to partly localize to the Golgi apparatus, endoplasmic reticulum and Spitzenkörper. Furthermore, we found that FgRab1 inactivation blocks not only the transport of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane but also the fusion of endocytic vesicles with their target membranes and general autophagy. In summary, our results indicate that FgRab1 plays vital roles in vegetative growth, conidiogenesis, pathogenicity, autophagy, vesicle fusion and trafficking in F. graminearum.

Arabidopsis RAB8A, RAB8B and RAB8D Proteins Interact with Several RTNLB Proteins and are Involved in the Agrobacterium tumefaciens Infection Process.

Arabidopsis thaliana small GTP-binding proteins, AtRAB8s, associate with the endomembrane system and modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways. There are five members in Arabidopsis, namely AtRAB8A-8E. Yeast two-hybrid assays, bimolecular fluorescence complementation assays and glutathione-S-transferase pull-down assays showed that RAB8A, 8B and 8D interacted with several membrane-associated reticulon-like (AtRTNLB) proteins in yeast, plant cells and in vitro. Furthermore, RAB8A, 8B and 8D proteins showed interactions with the Agrobacterium tumefaciens virulence protein, VirB2, a component of a type IV secretion system (T4SS). A. tumefaciens uses a T4SS to transfer T-DNA and Virulence proteins to plants, which causes crown gall disease in plants. The Arabidopsis rab8A, rab8B and rab8D single mutants showed decreased levels of Agrobacterium-mediated root and seedling transformation, while the RAB8A, 8B and 8D overexpression transgenic Arabidopsis plants were hypersusceptible to A. tumefaciens and Pseudomonas syringae infections. RAB8A-8E transcripts accumulated differently in roots, rosette leaves, cauline leaves, inflorescence and flowers of wild-type plants. In summary, RAB8A, 8B and 8D interacted with several RTNLB proteins and participated in A. tumefaciens and P. syringae infection processes.

The GTPase Rab1 Is Required for NLRP3 Inflammasome Activation and Inflammatory Lung Injury.

Uncontrolled inflammatory response during sepsis predominantly contributes to the development of multiorgan failure and lethality. However, the cellular and molecular mechanisms for excessive production and release of proinflammatory cytokines are not clearly defined. In this study, we show the crucial role of the GTPase Ras-related protein in brain (Rab)1a in regulating the nucleotide binding domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and lung inflammatory injury. Expression of dominant negative Rab1 N124I plasmid in bone marrow-derived macrophages prevented the release of IL-1ß and IL-18, NLRP3 inflammasome activation, production of pro-IL-1ß and pro-IL-18, and attenuated TLR4 surface expression and NF-кB activation induced by bacterial LPS and ATP compared with control cells. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of Rab1a-inactivated macrophages by expression of Rab1 N124I plasmid dramatically reduced the release of IL-1ß and IL-18, neutrophil count in bronchoalveolar lavage fluid, and inflammatory lung injury. Rab1a activity was elevated in alveolar macrophages from septic patients and positively associated with severity of sepsis and respiratory dysfunction. Thus, inhibition of Rab1a activity in macrophages resulting in the suppression of NLRP3 inflammasome activation may be a promising target for the treatment of patients with sepsis.

Focus on the Small GTPase Rab1: A Key Player in the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α-σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies.

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells.

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 ß and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.

Long Noncoding RNA MNX1 antisense RNA 1 Exerts Oncogenic Functions in Bladder Cancer by Regulating miR-218-5p/RAB1A Axis.

LncRNA MNX1 antisense RNA 1 (MNX1-AS1) is significantly overexpressed in patients with bladder cancer, suggesting that it might be associated with bladder cancer. However, the molecular mechanism of MNX1-AS1 in bladder cancer remained indistinct. To illustrate the role of MNX1-AS1 in bladder cancer, the gain- and loss-of-function experiments were conducted in bladder cancer cells. Reduced expression of MNX1-AS1 could suppress cell proliferation, migration, invasion, and epithelial-mesenchymal transition in bladder cancer cells, whereas overexpression of MNX1-AS1 resulted in the opposite effects. Mechanistic analysis demonstrated that miR-218-5p was a direct target of RAB1A. MNX1-AS1 could competitively bind to miR-218-5p to regulate RAB1A expression in bladder cancer cells. Furthermore, in vivo experiments revealed that reduced expression of MNX1-AS1 inhibited tumor growth and metastasis. Taken together, MNX1-AS1 functions as a sponge to miR-218-5p to modulate RAB1A expression in bladder cancer, which suggests that MNX1-AS1 might serve as a novel therapeutic target and a novel biomarker for metastasis and prognosis in bladder cancer. SIGNIFICANCE STATEMENT: Our study demonstrates that long noncoding RNA MNX1-AS1 promotes the initiation and progression of bladder cancer. MNX1-AS1 regulates RAB1A expression to promote proliferation, migration, invasion, and epithelial-mesenchymal transitions of bladder cancer cells via miR-218-5p, which contributes to the tumor growth and metastasis of bladder cancer. Collectively, these results suggest that MNX1-AS1 might serve as a potential biomarker for bladder cancer.

Micro and Martsolf syndromes in 34 new patients: Refining the phenotypic spectrum and further molecular insights.

Micro and Martsolf syndromes are rare clinically and genetically overlapping disorders caused by mutations in RAB3GAP1, RAB3GAP2, RAB18 and TBC1D20 genes. We describe 34 new patients, 27 with Micro and seven with Martsolf. Patients presented with the characteristic clinical manifestations of the two syndromes, including postnatal microcephaly, congenital cataracts, microphthalmia, optic atrophy, spasticity and intellectual disability. Brain imaging showed in the majority of cases polymicrogyria, thin corpus callosum, cortical atrophy, and white matter dysmyelination. Unusual additional findings were pectus excavatum (four patients), pectus carinatum (three patients), congenital heart disease (three patients) and bilateral calcification in basal ganglia (one patient). Mutational analysis of RAB3GAP1 and RAB3GAP2 revealed 21 mutations, including 14 novel variants. RAB3GAP1 mutations were identified in 22 patients with Micro, including a deletion of the entire gene in one patient. On the other hand, RAB3GAP2 mutations were identified in two patients with Micro and all Martsolf patients. Moreover, exome sequencing unraveled a TBC1D20 mutation in an additional family with Micro syndrome. Our results expand the phenotypic and mutational spectrum associated with Micro and Martsolf syndromes. Due to the overlapped severities and genetic basis of both syndromes, we suggest to be comprehended as one entity "Micro/Martsolf spectrum" or "RAB18 deficiency."

Rab1A knockdown represses proliferation and promotes apoptosis in gastric cancer cells by inhibition of mTOR/p70S6K pathway.

Rab1A, a member of the Ras-like protein in rat brain (Rab) family, acts as an oncogene in a variety of malignant tumors. Previous studies reported that Rab1A was highly expressed in GC tissues. However, the function and molecular mechanism of Rab1A in gastric cancer (GC) development remain far from being addressed. Rab1A mRNA and protein levels were detected by qRT-PCR and western blot, respectively. Cell proliferation was evaluated by CCK-8 and BrdU incorporation assays. Apoptosis was estimated by flow cytometry analysis and western blot analysis of B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), Bcl-2 associated X (Bax), and Bcl-2 homologous antagonist/killer (Bak) expression. Alteration of the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway was detected by western blot. We found that Rab1A expression at both mRNA and protein was upregulated in GC cells. Rab1A knockdown significantly inhibited cell proliferation and induced apoptosis in GC cells. Rab1A overexpression promoted proliferation, inhibited cisplatin-induced apoptosis, and increased xenograft growth. In addition, we found that Rab1A knockdown suppressed the mTOR/p70S6K pathway in GC cells. Moreover, activation of mTOR/p70S6K pathway by MHY1485 abolished the effects of Rab1A knockdown on cell proliferation and apoptosis. In conclusion, Rab1A knockdown repressed proliferation and promoted apoptosis in GC cells by inhibition of the mTOR/p70S6K pathway.

Martsolf syndrome with novel mutation in the TBC1D20 gene in a family from Iran.

Warburg Micro syndrome and Martsolf syndrome are phenotypically overlapping autosomal recessive conditions characterized by multiple organ abnormalities involving the ocular, nervous, and endocrine systems. Warburg Micro syndrome, the more severe of the two conditions, is caused by loss of function mutations in RAB3GAP1, RAB3GAP2, RAB18, and TBC1D20 genes, whereas Martsolf syndrome has been attributed to less damaging mutations in RAB3GAP1 and RAB3GAP2 genes. We report the clinical description and molecular characterization of a consanguineous Iranian family with two siblings, a male and a female, with dysmorphic features, bilateral congenital cataracts, optic nerve atrophy, congenital glaucoma, mild to moderate intellectual disability, seizures, hypogonadism, and mild osteoporosis. Spastic quadriplegia with contractures was observed in the male patient, while the female patient showed only mild hyperreflexia. Magnetic resonance imaging scans performed in the male patient showed a normal brain structure. Both siblings had neither microcephaly nor postnatal growth retardation. Whole exome sequencing identified a novel homozygous nonsense mutation [c.1060C>T; p.(Arg354Ter)] in the TBC1D20 gene in both siblings and confirmed the heterozygous carrier status of both parents. This report describes a novel mutation in the TBC1D20 gene in two Iranian patients with Martsolf syndrome, further extending the allelic heterogeneity and phenotypic spectrum of this rare condition. The genotype and phenotype of the patients are compared with those of Martsolf syndrome and Warburg Micro syndrome patients reported in the literature.

Spatial Cycling of Rab GTPase, Driven by the GTPase Cycle, Controls Rab's Subcellular Distribution.

Rab GTPases (>60 members in humans) function as master regulators of intracellular membrane trafficking. Correct and specific localization of Rab proteins is required for their function. How the distinct spatial distribution of Rab GTPases in the cell is regulated remains elusive. To globally assess the subcellular localization of Rab1, we determined kinetic parameters of two pathways that control the spatial cycles of Rab1, i.e., vesicular transport and GDP dissociation inhibitor (GDI)-mediated recycling. We demonstrate that the switching between GTP and GDP binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), GDI, and GDI displacement factor (GDF), is a major determinant of Rab1's ability to effectively cycle between cellular compartments and eventually its subcellular distribution. In silico perturbations of vesicular transport, GEFs, GAPs, GDI, and GDF using a mathematical model with simplified cellular geometries showed that these regulators play an important role in the subcellular distribution and activity of Rab1.

TBC1D20 deficiency induces Sertoli cell apoptosis by triggering irreversible endoplasmic reticulum stress in mice.

Male 'blind sterile' mice with the causative TBC1 domain family member 20 (TBC1D20) deficiency are infertile with excessive germ cell apoptosis and spermatogenesis arrest at the spermatid stage. Sertoli cells are characterised as 'nurse cells' essential for normal spermatogenesis, but the role and corresponding molecular mechanisms of TBC1D20 deficiency in Sertoli cells of mice are not clear to date. In the present study, the histopathology of the testis and Sertoli cell proliferation and apoptosis were determined, and the corresponding molecular mechanisms were investigated by western blotting. Our data showed that TBC1D20 exhibits a testis-abundant expression pattern, and its expression level is positively associated with spermatogenesis. TBC1D20 is assembled in the Golgi and endoplasmic reticulum and is widely expressed by various germ cell subtypes and Sertoli cells. TBC1D20 deficiency in Sertoli cells led to an excessive apoptosis ratio and G1/S arrest. The increased apoptosis of TBC1D20-deficient Sertoli cells resulted from caspase-12 activation. TBC1D20-deficient Sertoli cells had an abnormal Golgi-endoplasmic reticulum structure, which led to endoplasmic reticulum stress, resulting in cell cycle arrest and excessive apoptosis. It suggested that TBC1D20 deficiency triggers irreversible endoplasmic reticulum stress resulting in G1/S arrest and excessive apoptosis in TBC1D20-deficient Sertoli cells, and TBC1D20 deficiency in Sertoli cells may also contribute to the infertility phenotype in 'blind sterile' male mice.

Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance.

Translation elongation factor Tu (EF-Tu) is a conserved GTP-binding protein essential for protein translation in prokaryotes and in eukaryotic mitochondria and plastids. EF-Tu also has a GTP/GDP-independent chaperone activity that may function in acclimation to heat. Here, we report that the Arabidopsis (Arabidopsis thaliana) plastid EF-Tu, Rabe1b, rapidly becomes insoluble at temperatures as low as 35°C in vitro and 41°C in vivo, with more than 90% aggregation after 9 h at 45°C in vivo. Based on its established function in protein translation, heat-induced aggregation likely inactivates Rabe1b. To determine the effect of heat-induced aggregation, we isolated an Arabidopsis rabe1b knockdown mutant and discovered it to be highly compromised in heat tolerance. Overexpression of constitutive GTP- or GDP-bound mutant forms of Rabe1b in Arabidopsis and virus-induced silencing of Rabe1b in tomato (Solanum lycopersicum) also reduced heat tolerance. Compromised heat tolerance in the Arabidopsis rabe1b mutant and in the lines overexpressing constitutive GTP- or GDP-bound mutant Rabe1b proteins was associated with reduced plastid translation under heat stress. The Arabidopsis rabe1b mutant also showed compromised heat-induced expression of HsfA2 and its target genes. Constitutive overexpression of HsfA2 activated its target genes but only partially restored the heat tolerance of the rabe1b mutant. These results strongly suggest that the plastid protein EF-Tu is heat sensitive and acts as a critical limiting factor in plant heat stress responses, primarily functioning in plastid protein translation but also in protein folding and retrograde signaling of nuclear heat-responsive gene expression.

Both Peripheral Blood and Urinary miR-195-5p, miR-192-3p, miR-328-5p and Their Target Genes PPM1A, RAB1A and BRSK1 May Be Potential Biomarkers for Membranous Nephropathy.

BACKGROUND To identify noninvasive diagnostic biomarkers for membranous nephropathy (MN). MATERIAL AND METHODS The mRNA microarray datasets GSE73953 using peripheral blood mononuclear cells (PBMCs) of 8 membranous nephropathy patients and 2 control patients; and microRNAs (miRNA) microarray dataset GSE64306 using urine sediments of 4 membranous nephropathy patients and 6 control patients were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were respectively identified from PBMCs and urine sediments of membranous nephropathy patients, followed with functional enrichment analysis, protein-protein interaction (PPI) analysis, and miRNA-target gene analysis. Finally, the DEGs and the target genes of DEMs were overlapped to obtain crucial miRNA-mRNA interaction pairs for membranous nephropathy. RESULTS A total of 1246 DEGs were identified from PBMCs samples, among them upregulated CCL5 was found to be involved in the chemokine signaling pathway, and BAX was found to be apoptosis related; while downregulated PPM1A and CDK1 were associated with the MAPK signaling pathway and the p53 signaling pathway, respectively. The hub role of CDK1 (degree=18) and CCL5 (degree=12) were confirmed after protein-protein interaction network analysis in which CKD1 could interact with RAB1A. A total of 28 DEMs were identified in urine sediments. The 276 target genes of DEMs were involved in cell cycle arrest (PPM1A) and intracellular signal transduction (BRSK1). Thirteen genes were shared between the DEGs in PMBCs and the target genes of DEMs in urine sediments, but only hsa-miR-192-3p-RAB1A, hsa-miR-195-5p-PPM1A, and hsa-miR-328-5p-BRSK1 were negatively related in their expression level. CONCLUSIONS Both peripheral blood and urinary miR-195-5p, miR-192-3p, miR-328-5p, and their target genes PPM1A, RAB1A, and BRSK1 may be potential biomarkers for membranous nephropathy by participating in inflammation and apoptosis.