VTP-Identifier: Vesicular Transport Proteins Identification Based on PSSM Profiles and XGBoost

Vesicular transport proteins are related to many human diseases, and they threaten human health when they undergo pathological changes. Protein function prediction has been one of the most in-depth topics in bioinformatics. In this work, we developed a useful tool to identify vesicular transport proteins. Our strategy is to extract transition probability composition, autocovariance transformation and other information from the position-specific scoring matrix as feature vectors. EditedNearesNeighbours (ENN) is used to address the imbalance of the data set, and the Max-Relevance-Max-Distance (MRMD) algorithm is adopted to reduce the dimension of the feature vector. We used 5-fold cross-validation and independent test sets to evaluate our model. On the test set, VTP-Identifier presented a higher performance compared with GRU. The accuracy, Matthew’s correlation coefficient (MCC) and area under the ROC curve (AUC) were 83.6%, 0.531 and 0.873, respectively.

Polarized α-synuclein trafficking and transcytosis across Brain Endothelial Cells via Rab7-decorated carriers

Parkinsons disease is mainly caused by aggregation of alpha-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinsons disease and related synucleinopathies.

Flavivirus infections induce a Golgi stress response in vertebrate and mosquito cells

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3 and increased expression of pathway targeted genes. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on the Golgi complex imposed by virion and NS1 processing and secretion.

PKC-e Regulates Vesicle Delivery and Focal Exocytosis for Efficient IgG-mediated Phagocytosis

PKC-e is required for membrane addition during IgG-mediated phagocytosis; its role in this process is ill-defined. High resolution imaging revealed that PKC-e exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-e colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-e and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles are not delivered in macrophages from PKC-e null mice, or upon dissociation of the Golgi-associated pool of PKC-e, implicates Golgi-tethered PKC-e as a driver of Golgi-to-phagosome trafficking. Finally, we established that PKC-e's regulatory domain is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-e in focal exocytosis: its regulatory domain drives Golgi-derived vesicles to the phagosome while catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.

Centrosomal P4.1-associated protein (CPAP) positively regulates endocytic vesicular transport and lysosome targeting of EGFR

Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells. Here, we report a novel, positive regulatory influence for CPAP on endocytic vesicular transport (EVT) and lysosome targeting of internalized-cell surface receptor EGFR. We observed that higher CPAP levels cause an increase in the abundance of multi-vesicular body (MVB) and EGFR is detectable in CPAP-overexpression induced puncta. The surface and cellular levels of EGFR are higher under CPAP deficiency and lower under CPAP overexpression. While ligand-engagement induced internalization or routing of EGFR into early endosomes is not influenced by cellular levels of CPAP, we found that targeting of ligand-activated, internalized EGFR to lysosome is impacted by CPAP levels. Transport of ligand-bound EGFR from early endosome to late endosome/MVB and lysosome is diminished in CPAP-depleted cells. Moreover, CPAP depleted cells appear to show a diminished ability to form MVB structures upon EGFR activation. These observations suggest a positive regulatory effect of CPAP on EVT of ligand-bound EGFR-like cell surface receptors to MVB and lysosome. Overall, identification of a non-centriolar function of CPAP in endocytic trafficking provides new insights in understanding the non-canonical cellular functions of CPAP.

Flavivirus Infection Induce a Golgi Stress Response in Vertebrate and Mosquito Cells

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. Few examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on Golgi complex imposed by virion and NS1 processing and secretion.