Reversible ON- and OFF-switch receptors Clec4G and Rab1A reveal the hormetic effects of a pectin polysaccharide in Aralia elata (Miq.) Seem.

BACKGROUND: Numerous studies indicate that natural polysaccharides have immune-enhancing effects as a host defense potentiator. Few reports are available on hormetic effects of natural polysaccharides, and the underlying mechanisms remain unclear. PURPOSE: AELP-B6 (arabinose- and galactose-rich pectin polysaccharide) from Aralia elata (Miq.) Seem was taken as a case study to clarify the potential mechanism of hormetic effects of natural polysaccharides. METHODS: The pharmacodynamic effect of AELP-B6 was verified by constructing the CTX-immunosuppressive mouse model. The hormetic effects were explored by TMT-labeled proteomics, energy metabolism analysis, flow cytometry and western blot. The core-affinity target of AELP-B6 was determined by pull down, nanoLC-nanoESI+-MS, CETSA, immunoblot and SPR assay. The RAW264.7Clec4G-RFP and RAW264.7Rab1A-RFP cell lines were simultaneously constructed to determine the affinity difference between AELP-B6 and targets by confocal laser scanning live-cell imaging. Antibody blocking assays were further used to verify the mechanism of hormetic effects. RESULTS: AELP-B6 at low and medium doses may maintain the structural integrity of thymus and spleen, increase the concentrations of TNF-α, IFN-γ, IL-3 and IL-8, and alleviate CTX-induced reduction of immune cell viability in vivo. Proteomics and energy metabolism analysis revealed that AELP-B6 regulate HIF-1α-mediated metabolic programming, causing Warburg effects in macrophages. AELP-B6 at low and medium doses promoted the release of intracellular immune factors, and driving M1-like polarization of macrophages. As a contrast, AELP-B6 at high dose enhanced the expression levels of apoptosis related proteins, indicating activation of the intrinsic apoptotic cascade. Two highly expressed transmembrane proteins in macrophages, Clec4G and Rab1A, were identified as the primary binding targets of AELP-B6 which co-localized with the cell membrane and directly impacted with immune cell activation and apoptosis. AELP-B6 exhibits affinity differences with Clec4G and Rab1A, which is the key to the hormetic effects. CONCLUSION: We observed hormesis of natural polysaccharide (AELP-B6) for the first time, and AELP-B6 mediates the hormetic effects through two dose-related targets. Low dose of AELP-B6 targets Clec4G, thereby driving the M1-like polarization via regulating NF-κB signaling pathway and HIF-1α-mediated metabolic programming, whereas high dose of AELP-B6 targets Rab1A, leading to mitochondria-dependent apoptosis.

SNX32 is a host restriction factor that degrades African swine fever virus CP204L via the RAB1B-dependent autophagy pathway.

African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.

The Golgi complex of dopaminergic enteric neurons is fragmented in a hemiparkinsonian rat model.

Since gastrointestinal disorders are early consequences of Parkinson's disease (PD), this disease is clearly not restricted to the central nervous system (CNS), but also significantly affects the enteric nervous system (ENS). Large aggregates of the protein α-synuclein forming Lewy bodies, the prototypical cytopathological marker of this disease, have been observed in enteric nervous plexuses. However, their value in early prognosis is controversial. The Golgi complex (GC) of nigral neurons appears fragmented in Parkinson's disease, a characteristic common in most neurodegenerative diseases. In addition, the distribution and levels of regulatory proteins such as Rabs and SNAREs are altered, suggesting that PD is a membrane traffic-related pathology. Whether the GC of enteric dopaminergic neurons is affected by the disease has not yet been analyzed. In the present study, dopaminergic neurons in colon nervous plexuses behave as nigral neurons in a hemiparkinsonian rat model based on the injection of the toxin 6-OHDA. Their GCs are fragmented, and some regulatory proteins' distribution and expression levels are altered. The putative mechanisms of the transmission of the neurotoxin to the ENS are discussed. Our results support the possibility that GC structure and the level of some proteins, especially syntaxin 5, could be helpful as early indicators of the disease. RESEARCH HIGHLIGHTS: The Golgi complexes of enteric dopaminergic neurons appear fragmented in a Parkinson's disease rat model. Our results support the hypothesis that the Golgi complex structure and levels of Rab1 and syntaxin 5 could be helpful as early indicators of the disease.

Deubiquitinating enzyme PSMD7 promotes bladder cancer development: Involvement of RAB1A stabilization.

BACKGROUND: Proteasome 26S subunit, non-ATPase 7 (PSMD7) is a deubiquitinating enzyme that is involved in the stability of ubiquitinated proteins and participates in the development of multiple types of cancer. The roles of PSMD7 and its potential mechanisms in bladder cancer (BC) remain elusive. METHODS: In this study, we identified that PSMD7 was overexpressed in BC tissues based on gene expression omnibus (GEO) database and TNMplot web. To investigate the functional role of PSMD7, two BC cell lines, T24 and 5637, were selected. The cells were transfected with vectors containing short hairpin RNAs against PSMD7 or plasmids containing full-length PSMD7 to knockdown or overexpress PSMD7. RESULTS: Our results revealed that silencing PSMD7 inhibited cell proliferation, cycle progression, migration, invasion, and promoted cell apoptosis, whereas PSMD7 overexpression led to the opposite effects in the BC cells. Mechanically, PSMD7 influenced the protein expression but not the mRNA expression of the Ras-related protein Rab-1 A (RAB1A). PSMD7 combined with RAB1A and negatively regulated its ubiquitination, indicating that PSMD7 enhanced the stability of RAB1A through post-transcriptional modification. Moreover, the rescue experiment demonstrated that RAB1A was an important downstream effector molecule of PSMD7. Besides, the negative regulation of silencing PSMD7 on tumor growth was confirmed in mice. CONCLUSIONS: Our study substantiated a novel mechanism by which PSMD7 stabilized RAB1A to accelerate the progression of BC.

Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

Rab1A functioned as a binding protein involved in Macrobrachium rosenbergii Taihu virus infection.

Macrobrachium rosenbergii Taihu virus (MrTV) is a virulent pathogen that mainly threatens M. rosenbergii larvae. Rab proteins, which are essential for controlling intracellular membrane trafficking, are hijacked by multiple viruses to complete their life cycle. In this paper, we studied the function of M. rosenbergii Rab1A (MrRab1A) in the MrTV infection. Upon MrTV infection, the transcription level of MrRab1A was significantly up-regulated, indicating MrRab1A was a MrTV responsive gene and might be important for MrTV infection. Co-IP and co-localization assays revealed that MrRab1A could directly bind with MrTV and its capsid protein VP3. Moreover, the in vivo neutralization assay demonstrated that pre-incubation of MrTV with recombinant MrRab1A could partially block MrTV infection. These findings indicated that MrRab1A functioned as a virus-binding protein involved in MrTV infection, which shed new light on the mechanism of MrTV infection and provided a potential target for developing anti-MrTV therapies.

Grouper Rab1 inhibits nodovirus infection by affecting virus entry and host immune response.

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens, respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo, implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies.

LncRNA BANCR promotes oral squamous cell carcinoma progression via regulating Rab1A signaling.

BACKGROUND: Long non-coding RNA BRAF-activated non-protein coding RNA plays bidirectional roles in human cancers. However, function and molecular mechanism of BRAF-activated non-protein coding RNA in oral squamous cell carcinoma still need to clarify further. METHODS: Long non-coding RNA microarray assay, in situ hybridization staining, clinicopathological data analysis were performed to investigate expression pattern of BRAF-activated non-protein coding RNA in oral squamous cell carcinoma tissue samples. Constructing ectopically expressed BRAF-activated non-protein coding RNA in oral squamous cell carcinoma cells via plasmids or siRNAs, then changeable abilities of proliferation and motility of these cells were observed in vitro and in vivo. RNA-protein pulldown, RNA immunoprecipitation, and bioinformatics analyses were performed to explore potential pathways involved in BRAF-activated non-protein coding RNA-based regulation of malignant progression in oral squamous cell carcinoma. RESULTS: BRAF-activated non-protein coding RNA was identified upregulated in oral squamous cell carcinoma tissue and correlated with nodal metastasis and clinical severity of patients. Overexpressed BRAF-activated non-protein coding RNA increased percentage of 5-ethynyl-2'-deoxyuridine-positive cells, viability, migration, and invasion rates of oral squamous cell carcinoma cells, while silenced BRAF-activated non-protein coding RNA could observe weakened effects in vitro. Xenograft tumor formed by BRAF-activated non-protein coding RNA-overexpressed cells had bigger volume, faster growth rates, higher weight, and more Ki67+ cells. Pulmonary metastasis induced by BRAF-activated non-protein coding RNA-silenced cells had fewer colony nodes, Ki67+ cells, and CD31+ blood vessels. Furthermore, BRAF-activated non-protein coding RNA was mainly localized in nucleus of oral squamous cell carcinoma cells and bound Ras-associated binding 1A. Silencing Ras-associated binding 1A could damage mobile ability and phosphorylation levels of nuclear factor-κB in oral squamous cell carcinoma cells induced by overexpressing BRAF-activated non-protein coding RNA. Opposite trend was also observed. CONCLUSION: Acting as a promoter in oral squamous cell carcinoma metastasis, BRAF-activated non-protein coding RNA promotes oral squamous cell carcinoma cells proliferation and motility by regulating the BRAF-activated non-protein coding RNA/Ras-associated binding 1A complex, which activates nuclear factor-κB signaling pathway.

Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis.

Background: Recent therapeutic approaches have improved survival rate for women with breast cancer, but the survival rate for metastatic breast cancer is still low. Exosomes released by various cells are involved in all steps of breast cancer development. Methods: We established the multimodal imaging report expression in breast cancer cells with lentivirus vectors pGluc and pBirA to investigate the secreted exosomes. Comparative microRNA (miRNA) analysis was performed with miRNA qPCR array in mice with breast cancer lung metastasis. The co-immunoprecipitation and chromatin immunoprecipitation assays were used to identify the mechanism of miRNA sorting to exosomes. The potential therapeutic strategy using an anti-sorting antibody was used to investigate breast cancer lung metastasis. Results: We identified 26 high- and 32 low-expression level miRNAs in exosomes from metastasis compared to those from primary tumors and normal tissues. The tumor suppressors, including miR-200c and let-7a, were reduced in tumor tissues and metastasis but increased in the respective exosomes compared to normal tissues. Furthermore, the Ras-related protein (Rab1A) facilitated miR-200c sorting to exosomes circumventing the influence of tumor suppressor miR-200c on tumor cells, while the metastatic exosome cargo miR-200c inhibited F4/80+ macrophage immune response. Administration of anti-Rab1A antibody significantly repressed the trafficking of miR-200c to exosomes and breast cancer lung metastasis. Conclusion: Our study has identified a novel molecular mechanism for breast cancer lung metastasis mediated by exosome cargo miRNAs and provided a new therapeutic strategy for cancer immunotherapy.

The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering, and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 s half-life) colocalizing partially with the Golgi markers RAB1, Sed5, and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycle back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments, Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing a stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases the binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that, unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

Loss of BCAA catabolism enhances Rab1A-mTORC1 signaling activity and promotes tumor proliferation in NSCLC.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a leading cause of cancer death. Branched-chain amino acid (BCAA) homeostasis is important for normal physiological metabolism. Branched-chain keto acid dehydrogenase kinase (BCKDK) is a rate-limiting enzyme involved in BCAA degradation. BCAA metabolism has been highlighted in human cancers. The aberrant activation of mTORC1 has been implicated in tumor progression. Rab1A is a small GTPase, an activator of mTORC1, and an oncogene. This study aimed to reveal the specific role of BCKDK-BCAA-Rab1A-mTORC1 signaling in NSCLC. METHODS: We analyzed a cohort of 79 patients with NSCLC and 79 healthy controls. Plasma BCAA assays, immunohistochemistry, and network and pathway analyses were performed. The stable cell lines BCKDK-KD, BCKDK-OV A549, and H1299 were constructed. BCKDK, Rab1A, p-S6 and S6 were detected using western blotting to explore their molecular mechanisms of action in NSCLC. The effects of BCAA and BCKDK on the apoptosis and proliferation of H1299 cells were detected by cell function assays. RESULTS: We demonstrated that NSCLC was primarily involved in BCAA degradation. Therefore, combining BCAA, CEA, and Cyfra21-1 is clinically useful for treating NSCLC. We observed a significant increase in BCAA levels, downregulation of BCKDHA expression, and upregulation of BCKDK expression in NSCLC cells. BCKDK promotes proliferation and inhibits apoptosis in NSCLC cells, and we observed that BCKDK affected Rab1A and p-S6 in A549 and H1299 cells via BCAA modulation. Leucine affected Rab1A and p-S6 in A549 and H1299 cells and affected the apoptosis rate of H1299 cells. In conclusion, BCKDK enhances Rab1A-mTORC1 signaling and promotes tumor proliferation by suppressing BCAA catabolism in NSCLC, suggesting a new biomarker for the early diagnosis and identification of metabolism-based targeted approaches for patients with NSCLC.

CD83+ B cells alleviate uveitis through inhibiting DCs by sCD83.

Soluble CD83 (sCD83) exerts immunosuppressive functions in many autoimmune diseases, including experimental autoimmune uveitis (EAU), but the cells and mechanisms involved are unclear. This study showed that CD83+ B cells were the main sources of sCD83. They alleviated the symptoms of EAU and decreased the percentage of T cells and DCs in the eyes and lymph nodes. These CD83+ B cells decreased IL-1ß, IL-18 and IFN-γ secretion by DCs through sCD83. sCD83 interacted with GTPase Ras-related protein (Rab1a) in DCs to promote Rab1a accumulation in autolysosomes and inhibit mTORC1 phosphorylation and NLRP3 expression. Hence, CD83+ B cells play a regulatory role in EAU by secreting sCD83. The lack of regulation of CD83+ B cells might be an important factor leading to hyperimmune activation in patients with autoimmune uveitis. CD83+ B cells suppress activated DCs in uveitis, indicating the potential therapeutic role of CD83+ B cells in uveitis.

Circ_RBM23 knockdown suppresses chemoresistance, proliferation, migration and invasion of sorafenib-resistant HCC cells through miR-338-3p/RAB1B axis.

BACKGROUND: Circular RNA RNA-binding motif protein 23 (circ_RBM23; ID: hsa_circ_0000524) is a novel regulator in hepatocellular carcinoma (HCC). Herein, we planned to investigate its role in sorafenib resistance in HCC. METHOD: Levels of circ_RBM23, microRNA (miR)-338-3p, Ras-related GTPase-trafficking protein (RAB1B), Snail and E-cadherin were detected by real-time quantitative PCR and western blotting. Sorafenib resistant (SR) HCC cells (Huh7/SR and SK-HEP-1/SR) were established by acquisition of sorafenib resistance, and cell functions were measured by MTT assay, Edu assay, colony formation assay, apoptosis assay, transwell assay, and in vivo xenograft formation assay. Crosslink between miR-338-3p and circ_RBM23 or RAB1B was confirmed by bioinformatics analysis and dual-luciferase reporter assay. RESULTS: Circ_RBM23 upregulation was discovered in the tissues of SR patients and SR cells, which was accompanied with miR-338-3p downregulation and RAB1B upregulation. The 50% inhibitory concentration (IC50) of sorafenib in SR cells was greatly suppressed by interfering circ_RBM23 or reinforcing miR-338-3p, allied with this was the inhibition of EdU-positive cell rate, colony formation and migration/invasion abilities under sorafenib treatment, as well as the enhancement of apoptotic rate. Moreover, circ_RBM23 inhibition delayed tumor growth of Huh7/SR cells under sorfanib treatment in vivo. CONCLUSION: Circ_RBM23 promoted chemoresistance, malignant proliferation, migration and invasion of SR HCC cells by modulating miR-338-3p/RAB1B axis.

Inhibition of Rab1B Impairs Trafficking and Maturation of SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes cellular trafficking pathways to process its structural proteins and move them to the site of assembly. Nevertheless, the exact process of assembly and subcellular trafficking of SARS-CoV-2 proteins remains largely unknown. Here, we have identified and characterized Rab1B as an important host factor for the trafficking and maturation of the spike protein (S) after synthesis at the endoplasmic reticulum (ER). Using confocal microscopy, we showed that S and Rab1B substantially colocalized in compartments of the early secretory pathway. Co-expression of dominant-negative (DN) Rab1B N121I leads to an aberrant distribution of S into perinuclear spots after ectopic expression and in SARS-CoV-2-infected cells caused by either structural rearrangement of the ERGIC or Golgi or missing interaction between Rab1B and S. Western blot analyses revealed a complete loss of the mature, cleaved S2 subunit in cell lysates and culture supernatants upon co-expression of DN Rab1B N121I. In sum, our studies indicate that Rab1B is an important regulator of trafficking and maturation of SARS-CoV-2 S, which not only improves our understanding of the coronavirus replication cycle but also may have implications for the development of antiviral strategies.

ELMOD3-Rab1A-Flotillin2 cascade regulates lumen formation via vesicle trafficking in Ciona notochord.

Lumen development is a crucial phase in tubulogenesis, although its molecular mechanisms are largely unknown. In this study, we discovered an ELMO domain-containing 3 (ELMOD3), which belongs to ADP-ribosylation factor GTPase-activating protein family, was necessary to form the notochord lumen in Ciona larvae. We demonstrated that ELMOD3 interacted with lipid raft protein Flotillin2 and regulated its subcellular localization. The loss-of-function of Flotillin2 prevented notochord lumen formation. Furthermore, we found that ELMOD3 also interacted with Rab1A, which is the regulatory GTPase for vesicle trafficking and located at the notochord cell surface. Rab1A mutations arrested the lumen formation, phenocopying the loss-of-function of ELMOD3 and Flotillin2. Our findings further suggested that Rab1A interactions influenced Flotillin2 localization. We thus identified a unique pathway in which ELMOD3 interacted with Rab1A, which controlled the Flotillin2-mediated vesicle trafficking from cytoplasm to apical membrane, required for Ciona notochord lumen formation.

LINC00173 facilitates tumor progression by stimulating RAB1B-mediated PA2G4 and SDF4 secretion in nasopharyngeal carcinoma.

An increasing number of studies have found that long non-coding RNA (lncRNA) play important roles in driving the progression of nasopharyngeal carcinoma (NPC). Our microarray screening revealed that expression of the lncRNA long intergenic non-protein coding RNA 173 (LINC00173) was upregulated in NPC. However, its role and mechanism in NPC have not yet been elucidated. In this study, we demonstrate that high LINC00173 expression indicated a poor prognosis in NPC patients. Knockdown of LINC00173 significantly inhibited NPC cell proliferation, migration and invasion in vitro. Mechanistically, LINC00173 interacted and colocalized with Ras-related protein Rab-1B (RAB1B) in the cytoplasm, but the modulation of LINC00173 expression did not affect the expression of RAB1B at either the mRNA or protein levels. Instead, relying on the stimulation of RAB1B, LINC00173 could facilitate the extracellular secretion of proliferation-associated 2G4 (PA2G4) and stromal cell-derived factor 4 (SDF4; also known as 45-kDa calcium-binding protein) proteins, and knockdown of these proteins could reverse the NPC aggressive phenotype induced by LINC00173 overexpression. Moreover, in vivo LINC00173-knockdown models exhibited a marked slowdown in tumor growth and a significant reduction in lymph node and lung metastases. In summary, LINC00173 serves as a crucial driver for NPC progression, and the LINC00173-RAB1B-PA2G4/SDF4 axis might provide a potential therapeutic target for NPC patients.

Autophagy induced by Rab1a-ULK1 interaction promotes porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus from the Nidovirales order, continues to be a threat to the swine industry worldwide causing reproductive failure and respiratory disease in pigs. Previous studies have demonstrated that autophagy plays a positive role in PRRSV replication. However, its mechanism is less clearly understood. Herein, we report first that the protein level of Rab1a, a member of the Ras superfamily of GTPases, is upregulated during PRRSV infection. Subsequently, we demonstrate that Rab1a enhances PRRSV replication through an autophagy pathway as evidenced by knocking down the autophagy-related 7 (ATG7) gene, the key adaptor of autophagy. Importantly, we reveal that Rab1a interacts with ULK1 and promotes ULK1 phosphorylation dependent on its GTP-binding activity. These data indicate that PRRSV utilizes the Rab1a-ULK1 complex to initiate autophagy, which, in turn, benefits viral replication. These findings further highlight the interplay between PRRSV replication and the autophagy pathway, deepening our understanding of PRRSV infection.

Biochemical Structure and Function of TRAPP Complexes in the Cardiac System.

Trafficking protein particle (TRAPP) is well reported to play a role in the trafficking of protein products within the Golgi and endoplasmic reticulum. Dysfunction in TRAPP has been associated with disorders in the nervous and cardiovascular systems, but the majority of literature focuses on TRAPP function in the nervous system solely. Here, we highlight the known pathways of TRAPP and hypothesize potential impacts of TRAPP dysfunction on the cardiovascular system, particularly the role of TRAPP as a guanine-nucleotide exchange factor for Rab1 and Rab11. We also review the various cardiovascular phenotypes associated with changes in TRAPP complexes and their subunits.

A guide to selecting high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence.

Rab1 is a highly conserved small GTPase that exists in humans as two isoforms: Rab1A and Rab1B, sharing 92% sequence identity. These proteins regulate vesicle trafficking between the endoplasmic reticulum (ER) and Golgi and within the Golgi stacks. Rab1A and Rab1B may be oncogenes, as they are frequently dysregulated in various human cancers. Moreover, they contribute to the progression of Parkinson's disease. The availability of high-quality antibodies specific for Rab1A or Rab1B is essential to understand the distinct functions of these Rab1 proteins in both health and diseaseand to enhance the reproducibility of research involving these proteins. In this study, we characterized seven antibodies targeting Rab1A and five antibodies targeting Rab1B for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a much larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a valuable resource for the scientific community. While uses of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

SGOL2 promotes prostate cancer progression by inhibiting RAB1A ubiquitination.

Prostate cancer is the most prevalent genitourinary malignant cancer in men worldwide. Patients with prostate cancer who progress to castration-resistant prostate cancer (CRPC) or metastatic CRPC have significantly poorer survival. Advanced prostate cancer is a clinical challenge due to the lack of effective treatment strategies. In the field of oncology, SGOL2 was an emerging and differentially expressed molecule, which enhanced the proliferation of cell populations in vitro in our studies. Mass spectrum and Co-IP validated the interaction of SGOL2 and RAB1A in a protein-protein manner. We further investigated the role of SGOL2 in the regulatory mechanism of RAB1A in prostate cancer cell lines. Furthermore, SGOL2 regulated RAB1A expression by inhibiting its ubiquitination. Rescue Experiments demonstrated that SGOL2 promoted prostate cancer cell proliferation and migration by upregulating RAB1A expression. Finally, we found that SGOL2 and RAB1A may regulate the tumor microenvironment (TME) in prostate cancer. In conclusion, our findings concluded that SGOL2 stabilized RAB1A expression to promote prostate cancer development. Both of them were of great importance in TME modulation.