SUMOylated Golgin45 associates with PML-NB to transcriptionally regulate lipid metabolism genes during heat shock stress.

Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-ß2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.

Crosstalk between KDEL receptor and EGF receptor mediates cell proliferation and migration via STAT3 signaling.

Hostile microenvironment of cancer cells provoke a stressful condition for endoplasmic reticulum (ER) and stimulate the expression and secretion of ER chaperones, leading to tumorigenic effects. However, the molecular mechanism underlying these effects is largely unknown. In this study, we reveal that the last four residues of ER chaperones, which are recognized by KDEL receptor (KDELR), is required for cell proliferation and migration induced by secreted chaperones. By combining proximity-based mass spectrometry analysis, split venus imaging and membrane yeast two hybrid assay, we present that EGF receptor (EGFR) may be a co-receptor for KDELR on the surface. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon KDEL ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and mediates cell growth and migration. Taken together, our results suggest that KDELR serves as a bona fide cell surface receptor for secreted ER chaperones and transactivates EGFR-STAT3 signaling pathway.

KDEL Receptor Trafficking to the Plasma Membrane Is Regulated by ACBD3 and Rab4A-GTP.

KDEL receptor-1 maintains homeostasis in the early secretory pathway by capturing and retrieving ER chaperones to the ER during heavy secretory activity. Unexpectedly, a fraction of the receptor is also known to reside in the plasma membrane (PM), although it is largely unknown exactly how the KDEL receptor gets exported from the Golgi and travels to the PM. We have previously shown that a Golgi scaffolding protein (ACBD3) facilitates KDEL receptor localization at the Golgi via the regulating cargo wave-induced cAMP/PKA-dependent signaling pathway. Upon endocytosis, surface-expressed KDEL receptor undergoes highly complex itineraries through the Golgi and the endo-lysosomal compartments, where the endocytosed receptor utilizes Rab14A- and Rab11A-positive recycling endosomes and clathrin-decorated tubulovesicular carriers. In this study, we sought to investigate the mechanism through which the KDEL receptor gets exported from the Golgi en route to the PM. We report here that ACBD3 depletion results in greatly increased trafficking of KDEL receptor to the PM via Rab4A-positive tubular carriers emanating from the Golgi. Expression of constitutively activated Rab4A mutant (Q72L) increases the surface expression of KDEL receptor up to 2~3-fold, whereas Rab4A knockdown or the expression of GDP-locked Rab4A mutant (S27N) inhibits KDEL receptor targeting of the PM. Importantly, KDELR trafficking from the Golgi to the PM is independent of PKA- and Src kinase-mediated mechanisms. Taken together, these results reveal that ACBD3 and Rab4A play a key role in regulating KDEL receptor trafficking to the cell surface.

An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi.

KDEL receptor (KDELR) is a key protein that recycles escaped endoplasmic reticulum (ER) resident proteins from the Golgi apparatus back to the ER and maintains a dynamic balance between these two organelles in the early secretory pathway. Studies have shown that this retrograde transport pathway is partly regulated by two KDELR-interacting proteins, acyl-CoA-binding domain-containing 3 (ACBD3), and cyclic AMP-dependent protein kinase A (PKA). However, whether Golgi-localized ACBD3, which was first discovered as a PKA-anchoring protein in mitochondria, directly interacts with PKA at the Golgi and coordinates its signaling in Golgi-to-ER traffic has remained unclear. In this study, we showed that the GOLD domain of ACBD3 directly interacts with the regulatory subunit II (RII) of PKA and effectively recruits PKA holoenzyme to the Golgi. Forward trafficking of proteins from the ER triggers activation of PKA by releasing the catalytic subunit from RII. Furthermore, we determined that depletion of ACBD3 reduces the Golgi fraction of RII, resulting in moderate, but constitutive activation of PKA and KDELR retrograde transport, independent of cargo influx from the ER. Taken together, these data demonstrate that ACBD3 coordinates the protein secretory pathway at the Golgi by facilitating KDELR/PKA-containing protein complex formation.

Retro-2 alters Golgi structure.

Retro-2 directly interacts with an ER exit site protein, Sec16A, inhibiting ER exit of a Golgi tSNARE, Syntaxin5, which results in rapid re-distribution of Syntaxin5 to the ER. Recently, it was shown that SARS-CoV-2 infection disrupts the Golgi apparatus within 6-12 h, while its replication was effectively inhibited by Retro-2 in cultured human lung cells. Yet, exactly how Retro-2 may influence ultrastructure of the Golgi apparatus have not been thoroughly investigated. In this study, we characterized the effect of Retro-2 treatment on ultrastructure of the Golgi apparatus using electron microscopy and EM tomography. Our initial results on protein secretion showed that Retro-2 treatment does not significantly influence secretion of either small or large cargos. Ultra-structural study of the Golgi, however, revealed rapid accumulation of COPI-like vesicular profiles in the perinuclear area and a partial disassembly of the Golgi stack under electron microscope within 3-5 h, suggesting altered Golgi organization in these cells. Retro-2 treatment in cells depleted of GRASP65/55, the two well-known Golgi structural proteins, induced complete and rapid disassembly of the Golgi into individual cisterna. Taken together, these results suggest that Retro-2 profoundly alters Golgi structure to a much greater extent than previously anticipated.

Tankyrase-1-mediated degradation of Golgin45 regulates glycosyltransferase trafficking and protein glycosylation in Rab2-GTP-dependent manner.

Altered glycosylation plays an important role during development and is also a hallmark of increased tumorigenicity and metastatic potentials of several cancers. We report here that Tankyrase-1 (TNKS1) controls protein glycosylation by Poly-ADP-ribosylation (PARylation) of a Golgi structural protein, Golgin45, at the Golgi. TNKS1 is a Golgi-localized peripheral membrane protein that plays various roles throughout the cell, ranging from telomere maintenance to Glut4 trafficking. Our study indicates that TNKS1 localization to the Golgi apparatus is mediated by Golgin45. TNKS1-dependent control of Golgin45 protein stability influences protein glycosylation, as shown by Glycomic analysis. Further, FRAP experiments indicated that Golgin45 protein level modulates Golgi glycosyltransferease trafficking in Rab2-GTP-dependent manner. Taken together, these results suggest that TNKS1-dependent regulation of Golgin45 may provide a molecular underpinning for altered glycosylation at the Golgi during development or oncogenic transformation.

ACBD3 modulates KDEL receptor interaction with PKA for its trafficking via tubulovesicular carrier.

BACKGROUND: KDEL receptor helps establish cellular equilibrium in the early secretory pathway by recycling leaked ER-chaperones to the ER during secretion of newly synthesized proteins. Studies have also shown that KDEL receptor may function as a signaling protein that orchestrates membrane flux through the secretory pathway. We have recently shown that KDEL receptor is also a cell surface receptor, which undergoes highly complex itinerary between trans-Golgi network and the plasma membranes via clathrin-mediated transport carriers. Ironically, however, it is still largely unknown how KDEL receptor is distributed to the Golgi at steady state, since its initial discovery in late 1980s. RESULTS: We used a proximity-based in vivo tagging strategy to further dissect mechanisms of KDEL receptor trafficking. Our new results reveal that ACBD3 may be a key protein that regulates KDEL receptor trafficking via modulation of Arf1-dependent tubule formation. We demonstrate that ACBD3 directly interact with KDEL receptor and form a functionally distinct protein complex in ArfGAPs-independent manner. Depletion of ACBD3 results in re-localization of KDEL receptor to the ER by inducing accelerated retrograde trafficking of KDEL receptor. Importantly, this is caused by specifically altering KDEL receptor interaction with Protein Kinase A and Arf1/ArfGAP1, eventually leading to increased Arf1-GTP-dependent tubular carrier formation at the Golgi. CONCLUSIONS: These results suggest that ACBD3 may function as a negative regulator of PKA activity on KDEL receptor, thereby restricting its retrograde trafficking in the absence of KDEL ligand binding. Since ACBD3 was originally identified as PAP7, a PBR/PKA-interacting protein at the Golgi/mitochondria, we propose that Golgi-localization of KDEL receptor is likely to be controlled by its interaction with ACBD3/PKA complex at steady state, providing a novel insight for establishment of cellular homeostasis in the early secretory pathway.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

Proximity proteomics identifies novel function of Rab14 in trafficking of Ebola virus matrix protein VP40.

Ebola virus is a member of Filoviridae family of viruses that causes fetal hemorrhagic fever in human. Matrix protein VP40 of the Ebola virus is involved in multiple stages of viral maturation processes. In order to fully understand the interacting partners of VP40 in host cells, we applied proximity-dependent biotin-identification (BioID) approach to systematically screen for potential proteins at different time points of VP40 expression. By immunoprecipitation and subsequent proteomics analysis, we found over 100 candidate proteins with various cellular components and molecular functions. Among them, we identified Rab14 GTPase that appears to function at the late stage of VP40 expression. Imaging studies demonstrated that VP40 and Rab14 have substantial colocalization when expressed in HeLa cells. Overexpression of the dominant-negative Rab14(S25N) diminished the plasma membrane (PM) localization of VP40. In addition, we found that secreted VP40 protein can be endocytosed into Rab14 positive compartments. In summary, our study provides evidence that Rab14 is a novel regulator of the intracellular trafficking of Ebola virus matrix protein VP40 in HeLa cells.

Golgin45-Syntaxin5 Interaction Contributes to Structural Integrity of the Golgi Stack.

The unique stacked morphology of the Golgi apparatus had been a topic of intense investigation among the cell biologists over the years. We had previously shown that the two Golgin tethers (GM130 and Golgin45) could, to a large degree, functionally substitute for GRASP-type Golgi stacking proteins to sustain normal Golgi morphology and function in GRASP65/55-double depleted HeLa cells. However, compared to well-studied GM130, the exact role of Golgin45 in Golgi structure remains poorly understood. In this study, we aimed to further characterize the functional role of Golgin45 in Golgi structure and identified Golgin45 as a novel Syntaxin5-binding protein. Based primarily on a sequence homology between Golgin45 and GM130, we found that a leucine zipper-like motif in the central coiled-coil region of Golgin45 appears to serve as a Syntaxin5 binding domain. Mutagenesis study of this conserved domain in Golgin45 showed that a point mutation (D171A) can abrogate the interaction between Golgin45 and Syntaxin5 in pull-down assays using recombinant proteins, whereas this mutant Golgin45 binding to Rab2-GTP was unaffected in vitro. Strikingly, exogenous expression of this Syntaxin5 binding deficient mutant (D171A) of Golgin45 in HeLa cells resulted in frequent intercisternal fusion among neighboring Golgi cisterna, as readily observed by EM and EM tomography. Further, double depletion of the two Syntaxin5-binding Golgin tethers also led to significant intercisternal fusion, while double depletion of GRASP65/55 didn't lead to this phenotype. These results suggest that certain tether-SNARE interaction within Golgi stack may play a role in inhibiting intercisternal fusion among neighboring cisternae, thereby contributing to structural integrity of the Golgi stack.

Acyl-CoA-Binding Domain-Containing 3 (ACBD3; PAP7; GCP60): A Multi-Functional Membrane Domain Organizer.

Acyl-CoA-binding domain-containing 3 (ACBD3) is a multi-functional scaffolding protein, which has been associated with a diverse array of cellular functions, including steroidogenesis, embryogenesis, neurogenesis, Huntington's disease (HD), membrane trafficking, and viral/bacterial proliferation in infected host cells. In this review, we aim to give a timely overview of recent findings on this protein, including its emerging role in membrane domain organization at the Golgi and the mitochondria. We hope that this review provides readers with useful insights on how ACBD3 may contribute to membrane domain organization along the secretory pathway and on the cytoplasmic surface of intracellular organelles, which influence many important physiological and pathophysiological processes in mammalian cells.

ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b-GAP.

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.

Polymeric immunoglobulin receptor promotes tumor growth in hepatocellular carcinoma.

Deregulation of the immune system is believed to contribute to cancer malignancy, which has led to recent therapeutic breakthroughs facilitating antitumor immunity. In a malignant setting, immunoglobulin receptors, which are fundamental components of the human immune system, fulfill paradoxical roles in cancer pathogenesis. This study describes a previously unrecognized pro-oncogenic function of polymeric immunoglobulin receptor (pIgR) in the promotion of cell transformation and proliferation. Mechanistically, pIgR overexpression is associated with YES proto-oncogene 1, Src family tyrosine kinase (Yes) activation, which is required for pIgR-induced oncogenic growth. Specifically, pIgR activates the Yes-DNAX-activating protein of 12 kDa-spleen tyrosine kinase-Rac1/CDC42-MEK (extracellular signal-regulated kinase kinase)/ERK (extracellular signal-regulated kinase) cascade in an immunoreceptor tyrosine-based activating motif (ITAM)-dependent manner to promote cell transformation and tumor growth, although pIgR itself does not contain an ITAM sequence. Additionally, the combination of pIgR and phosphorylated Yes (p-Yes) levels serves as a prognostic biomarker for hepatitis B surface antigen-positive and early-stage hepatocellular carcinoma (HCC) patients. Moreover, pharmacological targeting of MEK/ERK or Yes represents a therapeutic option for the subgroup of patients with pIgR/p-Yes-positive HCC based on our results with both cancer cell-line-based xenografts and primary patient-derived xenografts. CONCLUSION: Our findings reveal the molecular mechanism by which pIgR promotes cancer malignancy, suggest the clinical potential of targeting this pathway in HCC, and provide new insight into the oncogenic role of immunoglobulin receptors. (Hepatology 2017;65:1948-1962).

Design, synthesis and pharmacological evaluation of 2-(thiazol-2-amino)-4-arylaminopyrimidines as potent anaplastic lymphoma kinase (ALK) inhibitors.

A series of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) was developed by incorporation of a substituted 2-aminothiazole component as the C-2 substituent of the center pyrimidine core. Compound 5i showed highest potency of 12.4 nM against ALK and 24.1 nM against ALK gatekeeper mutation L1196M. Although only having moderate cellular potency in the SUP-M2 cells harboring NPM-ALK, compound 5i showed good kinase selectivity and dose-dependently inhibited phosphorylation of ALK and its down-stream signaling pathways.

The role of polymeric immunoglobulin receptor in inflammation-induced tumor metastasis of human hepatocellular carcinoma.

BACKGROUND: Expression of the polymeric immunoglobulin receptor (pIgR), a transporter of polymeric IgA and IgM, is commonly increased in response to viral or bacterial infections, linking innate and adaptive immunity. Abnormal expression of pIgR in cancer was also observed, but its clinical relevance remains uncertain. METHODS: A human hepatocellular carcinoma (HCC) tissue microarray (n = 254) was used to investigate the association between pIgR expression and early recurrence. An experimental lung metastasis model using severe combined immune-deficient mice was applied to determine the metastatic potential of Madin-Darby canine kidney (n = 5 mice per group) and SMMC-7721 (n = 12 mice per group) cells overexpressing pIgR vs control cells. RNA interference, immunoprecipitation, and immunoblotting were performed to investigate the potential role for pIgR in the induction of epithelial-mesenchymal transition (EMT). In vitro studies (co-immunoprecipitation, immunoblotting, and migration, invasion, and adhesion assays) were used to determine the mechanisms behind pIgR-mediated metastasis. All statistical tests were two-sided. RESULTS: High expression of pIgR was statistically significantly associated with early recurrence in early-stage HCC and in hepatitis B surface antigen-positive HCC patients (log-rank P = .02). Mice injected with pIgR-overexpressing cells had a statistically significantly higher number of lung metastases compared with respective control cells (Madin-Darby canine kidney cells: pIgR mean = 29.4 metastatic nodules per lung vs control mean = 0.0 metastatic nodules per lung, difference = 29.4 metastatic nodules per lung, 95% confidence interval = 13.0 to 45.8, P = .001; SMMC-7721 cells: pIgR mean = 10.4 metastatic nodules per lung vs control mean = 2.2 metastatic nodules per lung, difference = 8.2 metastatic nodules per lung, 95% confidence interval = 1.0 to 15.5, P = .03). Furthermore, high expression of pIgR was sufficient to induce EMT through activation of Smad signaling. CONCLUSIONS: pIgR plays a role in the induction of EMT. Our results identify pIgR as a potential link between hepatitis B virus-derived hepatitis and HCC metastasis and provide evidence in support of pIgR as a prognostic biomarker for HCC and a potential therapeutic target.