Anti-GAPDH Antikörper (A121579)

Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3G^KO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3G^KO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.

© 2019. Published by The Company of Biologists Ltd.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A_2A receptor (A_2A R) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A_2A R blockade directly in microglia. Here we show that blocking microglial A_2A R prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure-induced death. The A_2A R antagonist SCH 58261 or the knockdown of A_2A R expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A_2A R antagonist decreased microglia proliferation, as well as the expression and release of pro-inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A_2A R blockade recapitulated the effects of microglia depletion, suggesting that blocking A_2A R in microglia is able to control neurodegeneration in glaucoma-like conditions. Importantly, in human organotypic retinal cultures, A_2A R blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A_2A R as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

© 2019 The Authors. Glia published by Wiley Periodicals, Inc.

Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.

Microtubules and F-actin, and their associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes, myosin-Va and kinesin-1 have both been proposed as long-range centrifugal transporters moving melanosomes into the peripheral dendrites. Here, we investigated the role of kinesin-1 heavy chain (Kif5b) and its suggested targeting factor Rab1a in transport. We performed confocal microscopy and subcellular fractionation, but did not detect Kif5b or Rab1a on melanosomes. Meanwhile functional studies, using siRNA knockdown and dominant negative mutants, did not support a role for Kif5b or Rab1a in melanosome transport. To probe the potential of Kif5b to function in transport, we generated fusion proteins that target active Kif5b to melanosomes and tested their ability to rescue perinuclear clustering in myosin-Va-deficient cells. Expression of these chimeras, but not full-length Kif5b, dispersed melanosomes with similar efficiency to myosin-Va. Our data indicate that kinesin and microtubules can compensate for defects in myosin-Va and actin-based transport in mammals, but that endogenous Kif5b does not have an important role in transport of melanocytes due to its inefficient recruitment to melanosomes.

© 2017. Published by The Company of Biologists Ltd.

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.

© 2016 Encarnação et al.

Chaperone-Mediated Autophagy is a selective form of autophagy. Recently, the degradation of a newly identified CMA substrate, the HIF1A transcription factor, was found to be regulated by the ubiquitin ligase STUB1. In this study we show, for the first time, that K63 ubiquitination is necessary for CMA degradation of HIF1A in vitro and in vivo. Additionally, STUB1 mediates K63 linked ubiquitination of HIF1A. Our findings add a new regulatory step and increase the specificity of the molecular mechanism involved in CMA degradation of HIF1A, expanding the role of ubiquitination to yet another biological process, since the same mechanism might be applicable to other CMA substrates.

The thymus is the major site for normal and leukemic T-cell development. The dissection of the molecular determinants of T-cell survival and differentiation is paramount for the manipulation of healthy or transformed T cells in cancer (immuno)therapy. Casein kinase 2 (CK2) is a serine/threonine protein kinase whose anti-apoptotic functions have been described in various hematological and solid tumors. Here we disclose an unanticipated role of CK2 in healthy human thymocytes that is selective to the ?d T-cell lineage. ?d thymocytes display higher (and T-cell receptor inducible) CK2 activity than their aß counterparts, and are strikingly sensitive to death upon CK2 inhibition. Mechanistically, we show that CK2 regulates the pro-survival AKT signaling pathway in ?d thymocytes and, importantly, also in ?d T-cell acute lymphoblastic leukemia (T-ALL) cells. When compared with healthy thymocytes or leukemic aß T cells, ?d T-ALL cells show upregulated CK2 activity, potentiated by CD27 costimulation, and enhanced apoptosis upon CK2 blockade using the chemical inhibitor CX-4945. Critically, this results in inhibition of tumor growth in a xenograft model of human ?d T-ALL. These data identify CK2 as a novel survival determinant of both healthy and leukemic ?d T cells, and may thus greatly impact their therapeutic manipulation.

Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle.

© 2016. Published by The Company of Biologists Ltd.

Gap junctions (GJs) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct intercellular communication between eukaryotic cells. Although Cx43 has long been known to be a substrate for ubiquitination, the reversal of this modification by deubiquitylases (DUBs) has never been described. Here we report that the DUB-associated molecule with the SH3 domain of STAM (AMSH) interacts with Cx43 and mediates its deubiquitination. In this study, we demonstrate that Cx43 is modified with lysine 63-linked polyubiquitin chains and that these increase the interaction between Cx43 and AMSH. We also show that AMSH is recruited to GJ plaque sites at the plasma membrane, where it mediates the deubiquitination of Cx43. Using siRNA depletion or overexpression of a catalytically inactive mutant of AMSH, we show that by decreasing Cx43 deubiquitination, both the internalization and degradation rate of Cx43 are increased. Overall, these data strongly suggest that AMSH-mediated deubiquitination of Cx43 protects GJs from degradation.

© FASEB.

The Arf-like protein Arl13b has been implicated in ciliogenesis and Sonic hedgehog signaling. Furthermore, we have previously shown that it regulates endocytic recycling traffic and interacts with actin. Herein, we report that the non-muscle myosin heavy chain IIA, also known as Myh9, is an Arl13b effector. Moreover, we found that both proteins localized to circular dorsal ruffles (CDRs) induced by platelet-derived growth factor stimulation and are required for their formation. CDRs are ring-shaped actin-dependent structures formed on the dorsal cell surface and are involved in diverse processes, such as macropinocytosis, integrin recycling, internalization of receptor tyrosine kinases and cell migration. We found that Arl13b or Myh9 silencing impaired cell migration, suggesting that Arl13b is required for this function through the interaction with Myh9. Moreover, Arl13b silencing impaired neural crest cell migration in zebrafish embryos. Furthermore, we showed that Arl13b is required for the formation of CDRs in migrating cells. Thus, our results indicate a new role for Arl13b in actin cytoskeleton remodeling through the interaction with Myh9, by driving the formation of CDRs necessary for cell migration.

© 2014. Published by The Company of Biologists Ltd.

INTRODUCTION AND OBJECTIVES:

Cardiovascular disease is the leading cause of mortality and morbidity associated with diabetes. Although impairment of the cell response to hypoxia due to destabilization of the transcription factor hypoxia-inducible factor-1a (HIF-1a), which regulates the expression of genes that help cells to cope with low oxygen tension, has been implicated in diabetes-associated disease, the molecular mechanisms involved remain elusive. It is known that hyperglycemia leads to the enhanced production of methylglyoxal (MGO). Therefore, the main objective of this study was to establish whether MGO leads to the degradation of HIF-1a in cardiomyocytes subjected to hypoxia.

METHODS:

The mouse atrial cardiomyocyte cell line, HL-1, was exposed to chemical hypoxia with CoCl₂ in the absence or presence of MGO. Cell viability was assessed by MTT assay, and levels of HIF-1a and endogenous ubiquitin conjugates were determined by western blotting. Proteasome activity was analyzed using a specific chymotrypsin-like fluorogenic substrate.

RESULTS:

The results obtained indicate that MGO induces time- and dose-dependent degradation of HIF-1a accumulated under hypoxia. Additionally, we show that accumulation of endogenous ubiquitin conjugates in the presence of MGO is associated with decreased proteasome activity.

CONCLUSION:

Taken together, the results obtained in this study suggest that MGO compromises the ability of cells to adapt to low oxygen tensions, by stimulating the degradation of HIF-1a, likely contributing to the development of diabetes-associated cardiac dysfunction.

Copyright © 2017 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L.U. All rights reserved.