Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Loss of RAB25 Cooperates with Oncogenes in the Transformation of Human Mammary Epithelial Cells (HMECs) to Give Rise to Claudin-Low Tumors.

The loss of RAB25 expression-RAS superfamily of GTPase characteristic of numerous breast cancers-corresponds with H-RAS point mutations, particularly in triple-negative breast cancers (TNBC), a subtype associated with a poor prognosis. To address the poorly understood factors dictating the progression of TNBC tumors, we examine the cooperative effects that loss of RAB25 expression in human mammary epithelial cell (HMEC) lines with H-RAS mutations confers in tumorigenesis. HMECs were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, a catalytic subunit of the telomerase enzyme. We found that with the loss of RAB25 and overexpression of mutant H-RAS61L, immortal HMECs transformed toward anchorage-independent growth and acquired an increased ability to migrate. Furthermore, cells express low CD24, high CD44, and low claudin levels, indicating stem-like properties upon transformation. Besides, loss of RAB25 and overexpression of H-RAS61L resulted in increased expression of transcription factors Snail and Slug that drive these cells to lose E-cadherin and undergo epithelial-mesenchymal transition (EMT). This study confirms that loss of RAB25 and overexpression of mutant H-RAS can drive HMECs toward a mesenchymal stem-like state. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker of the claudin-low type of TNBC.

Pseudorabies virus hijacks the Rab6 protein to promote viral assembly and egress.

Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.

Multi-tiered actions of Legionella effectors to modulate host Rab10 dynamics.

Rab GTPases are representative targets of manipulation by intracellular bacterial pathogens for hijacking membrane trafficking. Legionella pneumophila recruits many Rab GTPases to its vacuole and exploits their activities. Here, we found that infection-associated regulation of Rab10 dynamics involves ubiquitin signaling cascades mediated by the SidE and SidC families of Legionella ubiquitin ligases. Phosphoribosyl-ubiquitination of Rab10 catalyzed by the SidE ligases is crucial for its recruitment to the bacterial vacuole. SdcB, the previously uncharacterized SidC-family effector, resides on the vacuole and contributes to retention of Rab10 at the late stages of infection. We further identified MavC as a negative regulator of SdcB. By the transglutaminase activity, MavC crosslinks ubiquitin to SdcB and suppresses its function, resulting in elimination of Rab10 from the vacuole. These results demonstrate that the orchestrated actions of many L. pneumophila effectors fine-tune the dynamics of Rab10 during infection.

Legionella pneumophila exploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7.

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.

IL-33/NF-κB/ST2L/Rab37 positive-feedback loop promotes M2 macrophage to limit chemotherapeutic efficacy in lung cancer.

IL-33 is a danger signal that binds to its receptor ST2L to promote tumor progression. This study identifies the IL-33/ST2L positive-feedback loop and the trafficking of ST2L membrane presentation in macrophages that contribute to lung tumor progression. Mechanistically, IL-33 induces ST2L upregulation by activating NF-κB, which binds to the promoter region of the ST2L gene. Moreover, Rab37, a small GTPase involved in membrane trafficking, mediates ST2L trafficking to the plasma membrane of M2 macrophages. This IL-33/NF-κB/ST2L/Rab37 axis promotes positive-feedback loops that enhance ST2L expression and membrane trafficking in M2 macrophages. Notably, neutralizing antibodies against IL-33 or ST2L block NF-κB activity, suppress M2 macrophage polarization, and synergistically inhibit tumor growth when combined with cisplatin treatment in vitro/vivo. Clinically, Rab37+/ST2L+/CD206+ tumor-infiltrating M2 macrophages correlate with advanced-stage lung cancer patients with poor response to chemotherapy. These findings unveil a positive-feedback mechanism and provide a basis for IL-33/ST2L-targeting therapy for cancer.

The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.

Function and regulation of Rab GTPases in cancers.

The Rab small GTPases are characterized by the distinct intracellular localization and modulate various endocytic, transcytic and exocytic transport pathways. Rab proteins function as scaffolds that connect signaling pathways and intracellular membrane trafficking processes through the recruitment of effectors, such as tethering factors, phosphatases, motors and kinases. In different cancers, Rabs play as either an onco-protein or a tumor suppressor role, highly dependending on the context. The molecular mechanistic research has revealed that Rab proteins are involved in cancer progression through influences on migration, invasion, metabolism, exosome secretion, autophagy, and drug resistance of cancer cells. Therefore, targeting Rab GTPases to recover the dysregulated vesicle transport systems may provide potential strategy to restrain cancer progression. In this review, we discuss the regulation of Rab protein level and activity in modulating pathways involved in tumor progression, and propose that Rab proteins may serve as a prognostic factor in different cancers.

Rab30 facilitates lipid homeostasis during fasting.

To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2L-/-) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2L-/- mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

TBC9, an essential TBC-domain protein, regulates early vesicular transport and IMC formation in Toxoplasma gondii.

Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle trafficking system, which includes Rab GTPases and their regulators, to assure the biogenesis and secretory of the organelles. Here we exploit the model apicomplexan organism Toxoplasma gondii that encodes a family of Rab GTPase Activating Proteins, TBC (Tre-2/Bub2/Cdc16) domain-containing proteins. Functional profiling of these proteins in tachyzoites reveals that TBC9 is the only essential regulator, which is localized to the endoplasmic reticulum (ER) in T. gondii strains. Detailed analyses demonstrate that TBC9 is required for normal distribution of proteins targeting to the ER, and the Golgi apparatus in the parasite, as well as for the normal formation of daughter inner membrane complexes (IMCs). Pull-down assays show a strong protein interaction between TBC9 and specific Rab GTPases (Rab11A, Rab11B, and Rab2), supporting the role of TBC9 in daughter IMC formation and early vesicular transport. Thus, this study identifies the only essential TBC domain-containing protein TBC9 that regulates early vesicular transport and IMC formation in T. gondii and potentially in closely related protists.

Functional Divergence of the Paralog Salmonella Effector Proteins SopD and SopD2 and Their Contributions to Infection.

Salmonella enterica is a leading cause of bacterial food-borne illness in humans and is responsible for millions of cases annually. A critical strategy for the survival of this pathogen is the translocation of bacterial virulence factors termed effectors into host cells, which primarily function via protein-protein interactions with host proteins. The Salmonella genome encodes several paralogous effectors believed to have arisen from duplication events throughout the course of evolution. These paralogs can share structural similarities and enzymatic activities but have also demonstrated divergence in host cell targets or interaction partners and contributions to the intracellular lifecycle of Salmonella. The paralog effectors SopD and SopD2 share 63% amino acid sequence similarity and extensive structural homology yet have demonstrated divergence in secretion kinetics, intracellular localization, host targets, and roles in infection. SopD and SopD2 target host Rab GTPases, which represent critical regulators of intracellular trafficking that mediate diverse cellular functions. While SopD and SopD2 both manipulate Rab function, these paralogs display differences in Rab specificity, and the effectors have also evolved multiple mechanisms of action for GTPase manipulation. Here, we highlight this intriguing pair of paralog effectors in the context of host-pathogen interactions and discuss how this research has presented valuable insights into effector evolution.

A RabGAP negatively regulates plant autophagy and immune trafficking.

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

Activation of lipophagy is required for RAB7 to regulate ferroptosis in sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is the most common type of acute kidney injury (AKI), accompanied by elevated morbidity and mortality rates. This study investigated the mechanism by which lipid droplets (LDs) degraded via autophagy (lipophagy)required for RAB7 regulated ferroptosis in the pathogenesis of S-AKI. Here, we constructed the S-AKI model in vitro and in vivo to elucidate the potential relationship of lipophagy and ferroptosis, and we first confirmed that the activation of lipophagy promoted renal tubular epithelial cell ferroptosis and renal damage in S-AKI. The results showed that lipopolysaccharide (LPS) induced a marked increase in lipid peroxidation and ferroptosis, which were rescued by ferrstain-1 (Fer-1), an inhibitor of ferroptosis. In addition, LPS induced the remarkable activation of RAB7-mediated lipophagy. Importantly, silencing RAB7 alleviated LPS-induced lipid peroxidation and ferroptosis. Thus, the present study demonstrated the potential significant role of ferroptosis and lipophagy in sepsis-induced AKI, and contributed to better understanding of the pathogenesis and treatment targets of AKI.

Post-translational targeting of Rab35 by the effector IcsB of Shigella determines intracellular bacterial niche formation.

Escape from the bacterial-containing vacuole (BCV) is a key step of Shigella host cell invasion. Rab GTPases subverted to in situ-formed macropinosomes in the vicinity of the BCV have been shown to promote its rupture. The involvement of the BCV itself has remained unclear. We demonstrate that Rab35 is non-canonically entrapped at the BCV. Stimulated emission depletion imaging localizes Rab35 directly on the BCV membranes before vacuolar rupture. The bacterial effector IcsB, a lysine Nε-fatty acylase, is a key regulator of Rab35-BCV recruitment, and we show post-translational acylation of Rab35 by IcsB in its polybasic region. While Rab35 and IcsB are dispensable for the first step of BCV breakage, they are needed for the unwrapping of damaged BCV remnants from Shigella. This provides a framework for understanding Shigella invasion implicating re-localization of a Rab GTPase via its bacteria-dependent post-translational modification to support the mechanical unpeeling of the BCV.

The ras-related protein RAB22A interacts with hypoxia-inducible factor 1-alpha (HIF-1α) in MDA-MB-231 breast cancer cells in hypoxia.

BACKGROUND: Recent studies suggest that hypoxia-inducible factor 1-alpha (HIF-1α) and the small GTPase protein Ras-related protein Rab-22 A (RAB22A) may be colocalized in the cytoplasm and that as a conequence they may enhance the formation of microvesicles in breast cancer cells under hypoxia. Therefore, we sought to determine whether these two proteins are present in intracellular complexes in breast carcinoma cells. METHODS AND RESULTS: Evaluation using molecular docking indicated that HIF-1α and RAB22A interact with each other. Co-immunoprecipitation of endogenous or ectopically expressed HIF-1α and RAB22A proteins in MDA-MB-231 breast cancer cells or HEK-293T cells demonstrated that endogenous HIF-1α and RAB22A can form an intracellular complex; however, transiently expressed HIF-1α and RAB22A failed to interact. Investigating RAB22A and HIF-1α interactions in various cancer cell lines under hypoxia may shed light on their roles in cancer cell survival and progression through regulation of intracellular trafficking by HIF-1α under hypoxic conditions. CONCLUSIONS: Our study is the first to reveal the potential involvement of HIF-1α in intracellular trafficking through physical interactions with the small GTPase protein RAB22A. We discuss the implications of our work on the role of exosomes and microvesicles in tumor invasiveness.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

The vesicle trafficking gene, OsRab7, is critical for pollen development and male fertility in cytoplasmic male-sterility rice.

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.