A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

Rab26 controls secretory granule maturation and breakdown in Drosophila.

At the onset of Drosophila metamorphosis, plenty of secretory glue granules are released from salivary gland cells and the glue is deposited on the ventral side of the forming (pre)pupa to attach it to a dry surface. Prior to this, a poorly understood maturation process takes place during which secretory granules gradually grow via homotypic fusions, and their contents are reorganized. Here we show that the small GTPase Rab26 localizes to immature (smaller, non-acidic) glue granules and its presence prevents vesicle acidification. Rab26 mutation accelerates the maturation, acidification and release of these secretory vesicles as well as the lysosomal breakdown (crinophagy) of residual, non-released glue granules. Strikingly, loss of Mon1, an activator of the late endosomal and lysosomal fusion factor Rab7, results in Rab26 remaining associated even with the large glue granules and a concomitant defect in glue release, similar to the effects of Rab26 overexpression. Our data thus identify Rab26 as a key regulator of secretory vesicle maturation that promotes early steps (vesicle growth) and inhibits later steps (lysosomal transport, acidification, content reorganization, release, and breakdown), which is counteracted by Mon1.

Rab26 alleviates sepsis-induced immunosuppression as a master regulator of macrophage ferroptosis and polarization shift.

Macrophage dysfunction is a significant contributor to more than 70 % of sepsis-related deaths, specifically secondary bacterial infections, during the immunosuppression stage of sepsis. Nevertheless, the role of Rab26 in this context remains unclear. In this study, we observed a substantial decrease in Rab26 expression in macrophages during the immunosuppressive phase of sepsis, which was also found to be suppressed by high extracellular levels of HMGB1. During the progression of sepsis, Rab26 deficiency promotes a polarization shift from the M1 to the M2-like phenotype in macrophages, rendering them susceptible to ferroptosis. Subsequent experimentation has revealed that Rab26 deficiency facilitates the degradation of GPX4, thereby aggravating macrophage ferroptosis through the upregulation of levels of lipid ROS, MDA, and ferrous iron induced by RSL3, a ferroptosis inducer. Additionally, Rab26-deficient mice in the immunosuppressed phase of sepsis exhibit heightened susceptibility to secondary infections, leading to exacerbated lung tissue damage and increased mortality rate. Overall, these findings indicate that Rab26 plays a crucial role in sepsis-induced macrophage immunosuppression by regulating macrophage ferroptosis and polarization. Hence, it represents a potential novel target for sepsis therapy.

A GCC repeat in RAB26 undergoes natural selection in human and harbors divergent genotypes in late-onset Alzheimer's disease.

Although mainly located in genic regions and being mutation hotspots, intact blocks of CG-rich trinucleotide short tandem repeats (STRs) are largely overlooked with respect to their link with natural selection. The human RAB26 (member RAS oncogene family) directs synaptic and secretory vesicles into preautophagosomal structures, inhibition of which specifically disrupts axonal transport of degradative organelles and leads to an axonal dystrophy, resembling Alzheimer's disease (AD). Human RAB26 contains a GCC repeat in the top 1st percent in respect of length. Here we sequenced this STR in 441 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N = 216) and controls (N = 225). In both groups, the 12-repeat allele and the 12/12 genotype were predominantly abundant. We found excess of homozygosity for non-12 alleles in the NCD group (Mid-P exact = 0.027). Furthermore, divergent genotypes were detected that were specific to the NCD group (2.8% of genotypes) (Mid-P exact = 0.006) or controls (3.1% of genotypes) (Mid-P exact = 0.004). The patients harboring divergent genotypes received the diagnosis of AD. Based on the predominant abundance of the 12-repeat and 12/12 genotype in both groups, excess of non-12 homozygosity in the NCD group, and divergent genotypes across the NCD and control groups, we propose natural selection at this locus and link with late-onset AD. Our findings strengthen the hypothesis that a collection of rare genotypes unambiguously contribute to the pathogenesis of late-onset NCDs, such as AD.

Rab26 promotes macrophage phagocytosis through regulation of MFN2 trafficking to mitochondria.

Acute respiratory distress syndrome (ARDS) is an inflammatory disorder of the lungs caused by bacterial or viral infection. Timely phagocytosis and clearance of pathogens by macrophages are important in controlling inflammation and alleviating ARDS. However, the precise mechanism of macrophage phagocytosis remains to be explored. Here, we show that the expression of Rab26 is increased in Escherichia coli- or Pseudomonas aeruginosa-stimulated bone marrow-derived macrophages. Knocking out Rab26 reduced phagocytosis and bacterial clearance by macrophages. Rab26 interacts with mitochondrial fusion protein mitofusin-2 (MFN2) and affects mitochondrial reactive oxygen species generation by regulating MFN2 transport. The levels of MFN2 in mitochondria were reduced in Rab26-deficient bone marrow-derived macrophages, and the levels of mitochondrial reactive oxygen species and ATP were significantly decreased. Knocking down MFN2 using small interfering RNA resulted in decreased phagocytosis and killing ability of macrophages. Rab26 knockout reduced phagocytosis and bacterial clearance by macrophages in vivo, significantly increased inflammatory factors, aggravated lung tissue damage, and increased mortality in mice. Our results demonstrate that Rab26 regulates phagocytosis and clearance of bacteria by mediating the transport of MFN2 to mitochondria in macrophages, thus alleviating ARDS in mice and potentially in humans.

ROS induced the Rab26 promoter hypermethylation to promote cigarette smoking-induced airway epithelial inflammation of COPD through activation of MAPK signaling.

Cigarette smoking (CS) exposure-induced airway inflammatory responses drive the occurrence and development of emphysema and chronic obstructive pulmonary disease (COPD). However, its precise mechanisms have not been fully elucidated. In this study, we explore the role of Rab26 in CS exposure modulating the inflammatory response of airway epithelium and the novel mechanism of CS exposure regulation Rab26. These data showed that CS exposure and H2O2 (a type of ROS) suppressed the expression of Rab26 and increased the expression of DNMT3b in vivo and in vitro. GEO data analysis found the level of Rab26 was decreased in the lung tissue of COPD patients. CSE-induced ROS promoted DNA methylation of the Rab26 promoter and inhibited its promoter activity by elevating the DNMT3b level. Antioxidants N-Acetyl-l-cysteine (NAC), 5-Aza-2'-deoxycytidine (5-AZA) (DNA methylation inhibitor) and DNMT3B siRNA alleviated CSE's inhibitory effect on Rab26 expression in vitro. Importantly, NAC alleviated the improved expression of Rab26 and reduced DNMT3B expression, in the airway of smoking exposure as well as attenuated the inflammatory response in vivo. Overexpression of Rab26 attenuated CSE-induced production of inflammatory mediators through part inactivation of p38 and JNK MAPK. On the contrary, silencing Rab26 enhanced p38 and JNK activation and aggravated inflammatory response. These findings suggest that ROS-mediated Rab26 promoter hypermethylation is a critical step in cigarette smoking-induced airway epithelial inflammatory response. Restoring Rab26 in the airway epithelium might be a potential strategy for treating airway inflammation and COPD.

KLF4 targets RAB26 and decreases 5-FU resistance through inhibiting autophagy in colon cancer.

BACKGROUND: Accumulating studies demonstrated that resistance of colon cancer (CC) to 5-fluorouracil (5-FU) contributes to adverse prognosis. We investigated how Kruppel-like factor 4 (KLF4) affected 5-FU resistance and autophagy in CC cells. METHODS: KLF4 expression and its downstream target gene RAB26 in CC tissues was analyzed by bioinformatics analysis, and the effect of abnormal KLF4 expression on prognoses of CC patients was predicted. Luciferase reporter assay detected the targeted relationship between KLF4 and RAB26. The viability and apoptosis of CC cells were analyzed by CCK-8 and flow cytometry. The formation of intracellular autophagosomes was detected by confocal laser scanning microscopy and immunofluorescence staining. The mRNA and protein levels were assayed by qRT-PCR and western blot. A xenograft animal model was constructed to verify the function of KLF4. Rescue assay was employed to verify whether KLF4/RAB26 could affect 5-FU resistance in CC cells through autophagy. RESULTS: KLF4 and RAB26 were lowly expressed in CC. KLF4 correlated with patients' survival. KLF4 was down-regulated in 5-FU resistant CC cells. KLF4 overexpression suppressed the proliferation and 5-FU resistance of CC cells, and inhibited LC3 II/I expression and autophagosome formation. Autophagy activator Rapamycin or sh-RAB26 treatment reversed the impact of KLF4 overexpression on 5-FU resistance. In vivo assay verified that KLF4 inhibited 5-FU resistance in CC cells. Rescue experiments revealed that KLF4 targeted RAB26 to inhibit CC cell autophagy, resulting in decreasing the resistance to 5-FU. CONCLUSION: KLF4 strengthened the sensitivity of CC cells to 5-FU by targeting RAB26 to restrain autophagy pathway.

ATG9 resides on a unique population of small vesicles in presynaptic nerve terminals.

In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis.Abbreviations: AP: adaptor related protein complex: ATG2: autophagy related 2; ATG9: autophagy related 9; DNA PAINT: DNA-based point accumulation for imaging in nanoscale topography; DyMIN STED: dynamic minimum stimulated emission depletion; EL: endosome and lysosome; ER: endoplasmic reticulum; GA: Golgi apparatus; iBAQ: intensity based absolute quantification; LAMP: lysosomal-associated membrane protein; M6PR: mannose-6-phosphate receptor, cation dependent; Minflux: minimal photon fluxes; Mito: mitochondria; MS: mass spectrometry; PAS: phagophore assembly site; PM: plasma membrane; Px: peroxisome; RAB26: RAB26, member RAS oncogene family; RAB3A: RAB3A, member RAS oncogene family; RAB5A: RAB5A, member RAS oncogene family; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SVs: synaptic vesicles; SYP: synaptophysin; TGN: trans-Golgi network; TRAPP: transport protein particle; VTI1: vesicle transport through interaction with t-SNAREs.

RAB26 contributes to the progression of non-small cell lung cancer after being transcriptionally activated by SMAD3.

Non-small cell lung cancer (NSCLC) accounts for 85% of all cases of lung cancer, which constitutes the leading cause of cancer mortality. RAB26, a member of Rab GTPase superfamily, has been suggested to play a role in the tumorigenesis of NSCLC. The present work aimed to explore whether and how RAB26 contributed to the progression of NSCLC. NSCLC cell line A549 was transfection with short hairpin RNA (shRNA) or overexpression (Ov) vector to knockdown RAB26 or overexpress SMAD3, respectively. Then the malignant processes of A549 cells including proliferation, migration, invasion and apoptosis were evaluated by CCK-8, colony formation, wound-healing, transwell and TUNEL assays, respectively. Expression of proteins involved in these processes was measured by western blot. A549 xenograft mice model was established to confirm the effect of RAB26 silence on NSCLC progression in vivo. The relationship between RAB26 and SMAD3 was analyzed by bioinformatics and then verified by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. We found that silence of RAB26 inhibited the proliferation, migration and invasion but promoted apoptosis of A549 cells. In vivo studies revealed that the tumor growth of A549 xenograft was markedly suppressed upon RAB26 silence. Moreover, it was confirmed that SMAD3 bound to the promoter of RAB26 and enhance its expression. Finally, we observed that overexpression of SMAD3 significantly blocked the inhibitory effect of RAB26 silence on NSCLC progression. Collectively, RAB26 may contribute to the progression of NSCLC after being transcriptionally activated by SMAD3.

RBFOX2/GOLIM4 Splicing Axis Activates Vesicular Transport Pathway to Promote Nasopharyngeal Carcinogenesis.

20-30% of patients with nasopharyngeal carcinoma (NPC) develop distant metastasis or recurrence leading to poor survival, of which the underlying key molecular events have yet to be addressed. Here alternative splicing events in 85 NPC samples are profiled using transcriptome analysis and it is revealed that the long isoform of GOLIM4 (-L) with exon-7 is highly expressed in NPC and associated with poor prognosis. Lines of evidence demonstrate the pro-tumorigenic function of GOLIM4-L in NPC cells. It is further revealed that RBFOX2 binds to a GGAA motif in exon-7 and promotes its inclusion forming GOLIM4-L. RBFOX2 knockdown suppresses the tumorigenesis of NPC cells, phenocopying GOLIM4-L knockdown, which is significantly rescued by GOLIM4-L overexpression. High expression of RBFOX2 is correlated with the exon-7 inclusion of GOLIM4 in NPC biopsies and associated with worse prognosis. It is observed that RBFOX2 and GOLIM4 can influence vesicle-mediated transport through maintaining the organization of Golgi apparatus. Finally, it is revealed that RAB26 interacts with GOLIM4 and mediates its tumorigenic potentials in NPC cells. Taken together, the findings provide insights into how alternative splicing contributes to NPC development, by highlighting a functional link between GOLIM4-L and its splicing regulator RBFOX2 activating vesicle-mediated transport involving RAB26.

Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a condition with an imbalanced inflammatory response and delayed resolution of inflammation. Macrophage polarization plays an important role in inflammation and resolution. However, the mechanism of macrophage polarization in ALI/ARDS is not fully understood. We found that mice with lipopolysaccharide administration developed lung injury with the accumulation of extracellular cold-inducible RNA-binding protein (eCIRP) in the lungs. eCIRP, as a damage-associated molecular pattern (DAMP), inhibited M2 macrophage polarization, thereby tipping the balance toward inflammation rather than resolution. Anti-CIRP antibodies reversed such phenotypes. The levels of macrophage erythropoietin (EPO) receptor (EPOR) were reduced after eCIRP treatment. Myeloid-specific EPOR-deficient mice displayed restrained M2 macrophage polarization and impaired inflammation resolution. Mechanistically, eCIRP impaired Rab26, a member of Ras superfamilies of small G proteins, and reduced the transportation of surface EPOR, which resulted in macrophage polarization toward the M1 phenotype. Moreover, EPO treatment hardly promotes M2 polarization in Rab26 knockout (KO) macrophages through EPOR. Collectively, macrophage EPOR signaling is impaired by eCIRP through Rab26 during ALI/ARDS, leading to the restrained M2 macrophage polarization and delayed inflammation resolution. These findings identify a mechanism of persistent inflammation and a potential therapy during ALI/ARDS.

RAB26-dependent autophagy protects adherens junctional integrity in acute lung injury.

Microvascular barrier dysfunction is the central pathophysiological feature of acute lung injury (ALI). RAB26 is a newly identified small GTPase involved in the regulation of endothelial cell (EC) permeability. However, the mechanism behind this protection has not been clearly elucidated. Here we found that RAB26 promoted the integrity of adherens junctions (AJs) in a macroautophagy/autophagy-dependent manner in ALI. RAB26 is frequently downregulated in mouse lungs after LPS treatment. Mice lacking Rab26 exhibited phosphorylated SRC expression and increased CDH5/VE-cadherin phosphorylation, leading to AJ destruction. rab26-null mice showed further aggravation of the effects of endotoxin insult on lung vascular permeability and water content. Depletion of RAB26 resulted in upregulation of phosphorylated SRC, enhancement of CDH5 phosphorylation, and aggravation of CDH5 internalization, thereby weakening AJ integrity and endothelial barrier function in human pulmonary microvascular endothelial cells (HPMECs). RAB26 overexpression caused active interaction between SRC and the autophagy marker LC3-II and promoted degradation of phosphorylated SRC. Furthermore, RAB26 was involved in a direct and activation-dependent manner in autophagy induction through interaction with ATG16L1 in its GTP-bound form. These findings demonstrate that RAB26 exerts a protective effect on endothelial cell (EC) permeability, which is in part dependent on autophagic targeting of active SRC, and the resultant CDH5 dephosphorylation maintains AJ stabilization. Thus, RAB26-mediated autophagic targeting of phosphorylated SRC can maintain barrier integrity when flux through the RAB26-SRC pathway is protected. These findings suggest that activation of RAB26-SRC signaling provides a new therapeutic opportunity to prevent vascular leakage in ALI. ABBREVIATIONS: AJs: adherens junctions; ALI: acute lung injury; ARDS: acute respiratory distress syndrome; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG 16L1: autophagy related 16 like; 1 BALF: bronchoalveolar lavage fluidCQ: chloroquine; Ctrl: control; EC: endothelial cell; GFP: green fluorescent protein; HA-tagged; RAB26WT: HA-tagged wild-type; RAB26  HA-tagged; RAB26QL: HA-tagged; RAB26Q123LHA-tagged; RAB26NI: HA-tagged; RAB26N177IHPMECs: human pulmonary microvascular endothelial cells; H&E: hematoxylin & eosin; IgG: immunoglobulin; GIF: immunofluorescence; IP: immunoprecipitationi;. p.: intraperitoneal; LPS: lipopolysaccharide; PBS: phosphate-buffered salinesi; RNA: small interfering;RNASQSTM1/p62, sequestosome; 1TBS: Tris-buffered saline; VEGF: vascular endothelial growth factor; WB: western blot; WT: wild-type.

Role of Rab GTPases in the export trafficking of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute a superfamily of cell surface receptors that regulate a variety of cell functions. As the cell surface is the functional destination for most GPCRs, the cell surface targeting process represents a crucial checkpoint in controlling the functionality of the receptors. However, the molecular mechanisms underlying the cell surface delivery of newly synthesized GPCRs remain poorly understood. In this review, we will highlight the role of Rab GTPases in GPCR cell surface transport, particularly post-Golgi traffic, and discuss the underlying molecular mechanisms.

Regulation on Toll-like Receptor 4 and Cell Barrier Function by Rab26 siRNA-loaded DNA Nanovector in Pulmonary Microvascular Endothelial Cells.

The small GTPase Rab26 is involved in multiple processes, such as vesicle-mediated secretion and autophagy. However, the mechanisms and functions of Rab26 in the human pulmonary microvascular endothelial cells (HPMVECs) are not clear. In this study, we thoroughly investigated the role and novel mechanism of Rab26 in permeability and apoptosis of HPMVECs using a self-assembled Rab26 siRNA loaded DNA Y-motif nanoparticle (siRab26-DYM) and Rab26 adenovirus. We found that siRab26-DYM could be efficiently transfected into HPMVECs in a time- and dose-dependent manner. Importantly, the siRab26-DYM nanovector markedly aggravated the LPS-induced apoptosis and hyper-permeability of HPMVECs by promoting the nuclear translocation of Foxo1, and subsequent activation of Toll-like receptor 4 (TLR4) signal pathway. Overexpression of Rab26 by Rab26 adenoviruses partially inactivated LPS-induced TLR4 signaling pathway, suppressed the cell apoptosis and attenuated the hyperpermeability of HPMVECs. These results suggest that the permeability and apoptosis of HPMVECs can be modulated by manipulating Rab26 derived TLR4 signaling pathway, and that Rab26 can be potential therapeutic target for the treatment of vascular diseases related to endothelial barrier functions.

A Novel Human Rab26 cDNA Coloning,Expression and Its Endocytosis Enhancing Function / 生物化学与生物物理进展

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo-and endocytic pathways. Though there are many reports on Rab proteins, the function of these small proteins still remain in speculation. And no report has ever clarified the character of human Rab26. Here it was reported that a novel Rab protein Rab26 is membranous organelle related and in volved inendocytosis of HeLa cells. By using RT-PCR method a novel Rab26 cDNA full-length cDNA of Rab26 that is 1656 bp was identified.The cDNA sequence that at 1197 is 'A' other than 'G', while 'C' at 956 substitutes for 'T', and has 'GCC' insertion at 48 to 50 compared with published sequences. The complete open reading frame (ORF) is 771 bp in length encoding 256-residue protein with a calculated molecular mass of 27.9 ku (GenBank accession No.AY646153), rather than a shorter one with 190-amino acid residue as reported previously. GFP labeled full-length Rab26 expression showed that Rab26 was mainly sublocated in membranous organelles and could enhance endocytosis which means could took PE labeled protein as an endocytic tracer. RT-PCR analysis showed Rab26 was detected to express in several kinds of adenocarcinoma cell lines such as Acc2, AccM, SPC-A1 and HeLa cell lines, which indicated that Rab26 expression might be associated with some carcinomas.