Pancreatic β cell SERCA2 deficiency leads to impaired proinsulin processing and reduced ER to Golgi protein trafficking
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ABSTRACT:
PROVIDER: PRJNA855926 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19981503_1.fastq.gz | Fastqsanger.gz | |||
| SRR19981503_2.fastq.gz | Fastqsanger.gz | |||
| SRR19981504_1.fastq.gz | Fastqsanger.gz | |||
| SRR19981504_2.fastq.gz | Fastqsanger.gz | |||
| SRR19981505_1.fastq.gz | Fastqsanger.gz |
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Project description:Impaired proinsulin processing is observed in both type 1 and type 2 diabetes. We have previously shown that reductions in endoplasmic reticulum (ER) calcium (Ca2+) in the pancreatic β cell arising from impaired activity of the Sarcoendoplasmic Reticulum Ca2+ ATPase (SERCA) pump are associated with increased proinsulin secretion. However, the mechanisms responsible for reduced proinsulin processing in the context of SERCA2 deficiency remain incompletely understood. To test this, we developed mice with β cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance and reduced glucose-stimulated insulin secretion without evidence of impaired insulin sensitivity. ER Ca2+ levels in islets from βS2KO mice were significantly reduced, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2 and CPE. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Consistent with this, trafficking assays were performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Moreover, pancreas sections from βS2KO mice showed increased co-localization of proinsulin and ProPC2 in the early compartments of the secretory pathway. Taken together, these data suggest that loss of SERCA2 activity and ER Ca2+ loss in the pancreatic β cell leads to impaired proinsulin processing via reduced maturation and trafficking of proinsulin processing enzymes.
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Project description:Pancreatic cancer cells exploit vesicle trafficking proteins such as myoferlin to fuel tumor aggressiveness, yet the presence and function of myoferlin-dependent vesicles in cancer-associated fibroblasts (CAFs) remain unknown. By combining PDAC whole-tumor and single-cell transcriptomic analyses with immunohistochemistry and 2D/3D in vitro models, we link stromal myoferlin to tumor aggressiveness. We identify CAF-specific functions of myoferlin, as MYOF-depleted CAFs present reduced activity and impaired extracellular matrix (ECM) production. Analysis of intracellular vesicles identifies a TGFß-receptor 1 (TGFBR1) trafficking blockade at the ER/Golgi interface upon myoferlin depletion, leading to altered TGFBR1 activation, impaired signal transduction, loss of ECM production and reduced CAF contractility. The genetic depletion of myoferlin in the murine tumor stroma and the pharmacological targeting of myoferlin alike reduced tumor desmoplasia in orthotopic KPC mice. Overall, we propose TGFBR1 trafficking as innovative target to reprogram CAFs, control desmoplasia and tackle these aggressive features in pancreatic cancer.
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Project description:Androgen receptor (AR) plays an essential role in normal prostate development and prostate cancer (PCa) progression. To understand the role of AR at the single-cell level, we performed single-cell transcriptome analysis on PCa cells stimulated with androgen and antiandrogen to reconstruct the dynamic and direct AR transcriptional network. Our work reveals that androgen stimulates the ER and Golgi stress response , promoting secreting protein trafficking, and inhibiting cell apoptosis. Moreover, we identify an ER-to-Golgi protein vesicle-mediated transport gene signature essential for maximal androgen-mediated ER-Golgi trafficking, cell proliferation, and association with PCa prognosis and progression. Notably, we show that AR collaborates with CREB3L2, XXX, to coordinately promote ER-Golgi trafficking of Golgi enzyme Mannosidase II and PCa cell survival. Finally, we show the inhibition of the ER-Golgi transport process with Brefeldin A leads to tumor regression. Our study collectively reveals the heterogeneity of PCa cell transcriptional response to androgen stimulation, demonstrates a functional role for increased ER-Golgi trafficking process, and provides a mechanism for how the process is augmented in PCa as well as the potential of targeting may provide novel treatment strategies.
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Project description:Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs, thereby affecting their functions. We have previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in cell nuclei, suggesting that mRNA trafficking is impaired by expanded C9orf72-ALS repeats. To further characterize the process, in this work we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS repeats. Results from RNAseq profiling of nuclear and cytoplasmic RNA fractions obtained from cells expressing G4C231 repeats show a significant accumulation of mRNAs in the nuclei of G4C231 cells, further suggesting that nuclear retention of mRNAs is a major effect of C9orf72 expanded repeats expression. Interestingly, pathway analysis shows that mRNAs involved in ER-to-Golgi trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicate that the membrane trafficking route regulated by ARFGAP1, a GTPase-activating protein that associates with Golgi, is specifically affected by expanded C9orf72 repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), pointing to ER-to-Golgi vesicle-mediated transport as a target of C9orf72 toxicity.
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Project description:Targeting myoferlin in ER/Golgi vesicle trafficking reprograms cancer-associated fibroblasts in pancreatic cancer.
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Project description:The Golgi is the hub of the eukaryotic secretory pathway, trafficking proteins and lipids, as well as synthesizing complex sugars. Different biosynthetic reactions are associated with different compartments of its complex architecture. Although pre- and post-Golgi trafficking has been much studied, comparatively little is known about intra-Golgi organization. In this study, secretory vesicles and organelles were separated along an electrophoretic gradient at sub-Golgi resolution, presenting snapshots of the changing relative abundance of hundreds of resident and cargo proteins and glycans in transit through the ER, Golgi compartments and post-Golgi compartments. Furthermore, grouped features in migration profiles reveal the dominant intra-Golgi protein trafficking pathways, showing separate routes for cargo and different groups of resident proteins. As few structural characteristics of proteins or sequence motifs have been associated with specific regions of the Golgi stack, we also carried out a comparative analysis of the transmembrane regions of resident proteins associated with the main migratory profiles identifying the presence of charge amino acids adjacent to the transmembrane helix, exoplasmic Ser and Thr content and helix composition as likely contributors to protein sorting mechanisms.
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Project description:Flavivirus infection involves extensive remodeling of the endoplasmic reticulum (ER), which is key to both the replication of the viral RNA genome as well as the assembly and release of new virions. Yet, little is known about how viral proteins and host factors cooperatively facilitate such a vast transformation of the ER, and how this influences the different steps of the viral life cycle. In this study, we screened for host proteins that interact with the tick-borne encephalitis virus (TBEV) protein NS4B and found that the top candidates were coupled to trafficking between ER exit sites (ERES) and the Golgi. We characterized the role of ACBD3, one of the identified proteins, in flavivirus infection and show that it interacts with NS4B to promote infection across multiple flavivirus species. Using ACBD3 knockout cells, we found that the depletion of ACBD3 inhibited TBEV replication by preventing the trafficking of virions from the cell. We found that ACBD3 promotes flavivirus infection via a different mechanism than its previously described role in picornavirus infection. ACBD3 was enriched at modified ERES-Golgi contact sites to support virus replication. Therefore, we propose that ACBD3 promotes flavivirus replication by modifying the trafficking between the ERES and the Golgi to enable the release of new virions.
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Project description:Description: These date form the basis for supplementary table 2 and supplementary figure 6 of the manuscript "Modelling glycan processing reveals Golgi-enzyme homeostasis upon trafficking defects and cellular differentiation" by P Fisher, H Spencer, J Thomas-Oates, AJ Wood, D Ungar in Cell Reports
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Project description:The anterograde secretory pathway shapes the extracellular environment and the ability of cells to communicate, through the release of cytokines, growth factors, enzymes and extracellular matrix components, and through the regulation of the composition of the plasma membrane (PM). The underlying molecular machinery operates in a concerted and highly regulated manner and is frequently altered in cancer. The Golgi RabGAP protein TBC1D22B is overexpressed in breast cancer and predicts prognosis. Herein, deconvolution of the proximity-labeling and physical interactomes of TBC1D22B, reveals its role in the regulation of anterograde trafficking with liaisons with networks of proteins involved in cell adhesion, and in carbohydrate and lipid metabolism. The function of TBC1D22B in the anterograde pathway was formally demonstrated using the Retention Using Selective Hooks system which synchronizes transport of cargoes from the ER to the Golgi and the PM. TBC1D22B inhibits ER to Golgi transport in a GAP-dependent manner. RAB1B, a key RAB in the early secretory pathway, was found to be a substrate of the TBC1D22B GAP activity, and its silencing phenocopied TBC1D22B overexpression. Thus, TBC1D22B regulates ER to Golgi trafficking through its ability to work as a GAP for RAB1B.
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