The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport

Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human “ubiquitinome” using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes

Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein <i>via </i>the MVB/lysosomal pathway

© FASEB. Brain regions affected by Alzheimer disease (AD) displaywell-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction.Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment b (C99), generated by cleavage of APP by b-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement inADand the earliest initiator of synaptic plasticity and long-termmemory impairment. The aimof the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosomeformationorblock the fusionof autophagosomes to

Antiviral activity of an endogenous parvoviral element

Endogenous viral elements (EVEs) are genomic DNA sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express proteins with physiological roles in their host. Furthermore, some EVEs exhibit a protective role against exogenous viral infection in their host. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and although some of them contain ORFs, their function is unknown. We have shown that the locus EPV-Dependo.43-ODegus, an EPV with an intact ORF, is transcribed in Octodon degus (degu). Here we examine the antiviral activity of the protein encoded in this EPV, named DeRep. DeRep was produced in bacteria and used to generate antibodies that recognize DeRep in western blots of degu tissue. To test if DeRep could protect against exogenous parvovirus, we challenged cells with the minute virus of mice (MVM), a model autonomous parvovirus. We observed that MVM protein expression, DNA damage induced by replication, viral DNA, and cytopathic effects are reduced when DeRep is expressed in cells. The results of this study demonstrate that DeRep is expressed in degu and can inhibit parvovirus replication. This is the first time that an EPV has been shown to have antiviral activity against an exogenous virus

Puesta en marcha de un sistema de evaluación formativa multidisciplinar aplicado a asignaturas de Ingeniería Química y Nutrición en el Grado en Ciencia y Tecnología de los Alimentos

Ponencia presentada en: VIII Jornadas de Innovación Docente de la UBU, Burgos, 5 de abril de 2016, organizadas por el Instituto de Formación e Innovación Educativa-IFIE de la Universidad de Burgo

Retromer-containing tubules leaving early endosomes [4e. Honorable Mention]

Presentes the retromer is a multisubunit complex that mediates retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN). The CI-MPR binds newly-synthesized acid hydrolases at the TGN and carries them within clathrin-coated vesicles to endosomes, where the hydrolases are released for eventual transport to lysosomes. The retromer then functions to retrieve the unoccupied receptors to the TGN, where they engage in further cycles of acid hydrolase sorting. The mammalian retromer comprises two biogenetically distinct subcomplexes of tightly assembled subunits: a homodimer or heterodimer of SNX1 or SNX2 and a heterotrimer composed of Vps26, Vps29 and Vps35. This video shows a HeLa cell transiently expressing Vps29-GFP. By means of high-speed live-cell imaging the video shows the association of Vps29-GFP to both early endosomes and highly mobile vesicles. It also shows the retromer in tubular elements detaching from early endosomesComponente Curricular::Educação Superior::Ciências Biológicas::Morfologi

Retromer-containing tubules leaving early endosomes [4e. Honorable Mention]

The retromer is a multisubunit complex that mediates retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN). The CI-MPR binds newly-synthesized acid hydrolases at the TGN and carries them within clathrin-coated vesicles to endosomes, where the hydrolases are released for eventual transport to lysosomes. The retromer then functions to retrieve the unoccupied receptors to the TGN, where they engage in further cycles of acid hydrolase sorting. The mammalian retromer comprises two biogenetically distinct subcomplexes of tightly assembled subunits: a homodimer or heterodimer of SNX1 or SNX2 and a heterotrimer composed of Vps26, Vps29 and Vps35. This video shows a HeLa cell transiently expressing Vps29-GFP. By means of high-speed live-cell imaging the video shows the association of Vps29-GFP to both early endosomes and highly mobile vesicles. It also shows the retromer in tubular elements detaching from early endosomesComponente Curricular::Educação Superior::Ciências Biológicas::Biologia Gera

Traffic Jam: the dynamic behavior of a golgi matrix protein [4c. Honorable mention]

Componente Curricular::Educação Superior::Ciências Biológicas::Biologia GeralThis video shows an NRK cell stably expressing the Golgi matrix protein GRASP55-GFP. The Golgi apparatus in mammalian cells is made up of stacked cisternae organized in a ribbonlike structure within a ribosome-free Golgi matrix. The matrix is composed of coiled-coil proteins, many of which were first identified as autoantigens and termed Golgins. Golgi matrix proteins were thought to be stably associated with the matrix once synthesized, but increasing evidence suggests they are dynamic. By means of high-speed live-cell imaging the video shows the highly dynamic behavior of GRASP55-GFP-containing tubulo/vesicular structures moving in anterograde and retrograde fashion between ER exit sites and the Golgi apparatu