scholarly journals The E3 ubiquitin ligase MARCH2 regulates ERGIC3-dependent trafficking of secretory proteins

2019 ◽  
Vol 294 (28) ◽  
pp. 10900-10912 ◽  
Author(s):  
Wonjin Yoo ◽  
Eun-Bee Cho ◽  
Sungjoo Kim ◽  
Jong-Bok Yoon
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
E3 Ubiquitin Ligase ◽  
Secretory Proteins ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Lysine Residues ◽  
Subsequent Degradation ◽  
And Function

The E3 ubiquitin ligase membrane-associated ring-CH–type finger 2 (MARCH2) is known to be involved in intracellular vesicular trafficking, but its role in the early secretory pathway between the endoplasmic reticulum (ER) and Golgi compartments is largely unknown. Human ER–Golgi intermediate compartment protein 2 (ERGIC2) and ERGIC3 are orthologs of Erv41 and Erv46 in yeast, proteins that form a heteromeric complex, cycle between the ER and Golgi, and function as cargo receptors in both anterograde and retrograde protein trafficking. Here, we report that MARCH2 directs ubiquitination and subsequent degradation of ERGIC3 and that MARCH2 depletion increases endogenous ERGIC3 levels. We provide evidence that the lysine residues at positions 6 and 8 of ERGIC3 are the major sites of MARCH2-mediated ubiquitination. Of note, MARCH2 did not significantly decrease the levels of an ERGIC3 variant with lysine-to-arginine substitutions at residues 6 and 8. We also show that ERGIC3 binds to itself or to ERGIC2, whereas ERGIC2 is unable to interact with itself. Our results indicate that α1-antitrypsin and haptoglobin are likely to be cargo proteins of ERGIC3. We further observed that α1-antitrypsin and haptoglobin specifically bind to ERGIC3 and that ERGIC3 depletion decreases their secretion. Moreover, MARCH2 reduced secretion of α1-antitrypsin and haptoglobin, and coexpression of the ubiquitination-resistant ERGIC3 variant largely restored their secretion, suggesting that MARCH2-mediated ERGIC3 ubiquitination is the major cause of the decrease in trafficking of ERGIC3-binding secretory proteins. Our findings provide detailed insights into the regulation of the early secretory pathway by MARCH2 and into ERGIC3 function.

scholarly journals Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs

2020 ◽  
Vol 133 (19) ◽  
pp. jcs250100
Author(s):  
Simon Newstead ◽  
Francis Barr
Keyword(s):  
Secretory Pathway ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Retrograde Transport ◽  
Coated Vesicles ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Protein Localisation ◽  
And Function ◽  
Trafficking Receptor

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.

scholarly journals Commonly used trafficking blocks disrupt ARF1 activation and the localization and function of specific Golgi proteins

2018 ◽  
Vol 29 (8) ◽  
pp. 937-947 ◽  
Author(s):  
Catherine E. Gilbert ◽  
Elizabeth Sztul ◽  
Carolyn E. Machamer
Keyword(s):  
Protein Trafficking ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Small Gtpase ◽  
Cold Temperature ◽  
Nucleotide Exchange ◽  
Specific Subset ◽  
Nucleotide Exchange Factor ◽  
Cargo Proteins ◽  
And Function

ADP-ribosylation factor (ARF) proteins are key regulators of the secretory pathway. ARF1, through interacting with its effectors, regulates protein trafficking by facilitating numerous events at the Golgi. One unique ARF1 effector is golgin-160, which promotes the trafficking of only a specific subset of cargo proteins through the Golgi. While studying this role of golgin-160, we discovered that commonly used cold temperature blocks utilized to synchronize cargo trafficking (20 and 16°C) caused golgin-160 dispersal from Golgi membranes. Here, we show that the loss of golgin-160 localization correlates with a decrease in the levels of activated ARF1, and that golgin-160 dispersal can be prevented by expression of a GTP-locked ARF1 mutant. Overexpression of the ARF1 activator Golgi brefeldin A–resistant guanine nucleotide exchange factor 1 (GBF1) did not prevent golgin-160 dispersal, suggesting that GBF1 may be nonfunctional at lower temperatures. We further discovered that several other Golgi resident proteins had altered localization at lower temperatures, including proteins recruited by ARF-like GTPase 1 (ARL1), a small GTPase that also became dispersed in the cold. Although cold temperature blocks are useful for synchronizing cargo trafficking through the Golgi, our data indicate that caution must be taken when interpreting results from these assays.

scholarly journals Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

Open Biology ◽  
2014 ◽  
Vol 4 (3) ◽  
pp. 130172 ◽  
Author(s):  
Barbara Franke ◽  
Alexander Gasch ◽  
Dayté Rodriguez ◽  
Mohamed Chami ◽  
Muzamil M. Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Quaternary Structure ◽  
E3 Ubiquitin Ligase ◽  
Coiled Coil ◽  
Trim Protein ◽  
Muscle Catabolism ◽  
And Function ◽  
Helical Region ◽  
Structural Significance

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.

scholarly journals Structure and function of E3 ubiquitin ligase mindbomb RING domain

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Robert D Wardlow ◽  
Sung Hee Choi ◽  
Brian McMillan ◽  
Stephen C Blacklow
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structure And Function ◽  
Ring Domain ◽  
And Function

scholarly journals An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

1989 ◽  
Vol 109 (1) ◽  
pp. 17-34 ◽  
Author(s):  
P Rosa ◽  
U Weiss ◽  
R Pepperkok ◽  
W Ansorge ◽  
C Niehrs ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Intracellular Localization ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Double Labeling ◽  
Protein Protein Interaction ◽  
And Function ◽  
Secretogranin I

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Proteolysis-targeting chimeras mediate the degradation of bromodomain and extra-terminal domain proteins

2020 ◽  
Vol 12 (18) ◽  
pp. 1669-1683
Author(s):  
Yifei Yang ◽  
Zhenwei Wu ◽  
Pan Chen ◽  
Peiyuan Zheng ◽  
Huibin Zhang ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Side Effects ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Drug Discovery And Development ◽  
Terminal Domain ◽  
Super Enhancer ◽  
Bet Inhibitors ◽  
Subsequent Degradation ◽  
Bet Protein

Bromodomain and extra-terminal domain (BET) protein family plays an important role in regulating gene transcription preferentially at super-enhancer regions and has been involved with several types of cancers as a candidate. Up to now, there are 16 pan-BET inhibitors in clinical trials, however, most of them have undesirable off-target and side-effects. The proteolysis-targeting chimeras technology through a heterobifunctional molecule to link the target protein and E3 ubiquitin ligase, causes the target’s ubiquitination and subsequent degradation. By using this technology, the heterobifunctional small-molecule BET degraders can induce BET protein degradation. In this review, we discuss the advances in the drug discovery and development of BET-targeting proteolysis-targeting chimeras.

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

2000 ◽  
Author(s):  
Jens Rietdorf ◽  
David J. Stephens ◽  
Anthony Squire ◽  
Jeremy Simpson ◽  
David T. Shima ◽  
...  
Keyword(s):  
Cellular Structure ◽  
3D Imaging ◽  
Secretory Pathway ◽  
Living Cells ◽  
Structure And Function ◽  
Early Secretory Pathway ◽  
And Function

scholarly journals A VCP inhibitor substrate trapping approach (VISTA) enables proteomic profiling of endogenous ERAD substrates

2018 ◽  
Vol 29 (9) ◽  
pp. 1021-1030 ◽  
Author(s):  
Edmond Y. Huang ◽  
Milton To ◽  
Erica Tran ◽  
Lorraine T. Ador Dionisio ◽  
Hyejin J. Cho ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Carcinoma Cells ◽  
26S Proteasome ◽  
Proteomic Profiling ◽  
Aaa Atpase ◽  
Early Secretory Pathway ◽  
Lysine Residues ◽  
Discovery Method ◽  
Established Role ◽  
Human Hepatocellular Carcinoma Cells

Endoplasmic reticulum (ER)–associated degradation (ERAD) mediates the proteasomal clearance of proteins from the early secretory pathway. In this process, ubiquitinated substrates are extracted from membrane-embedded dislocation complexes by the AAA ATPase VCP and targeted to the cytosolic 26S proteasome. In addition to its well-established role in the degradation of misfolded proteins, ERAD also regulates the abundance of key proteins such as enzymes involved in cholesterol synthesis. However, due to the lack of generalizable methods, our understanding of the scope of proteins targeted by ERAD remains limited. To overcome this obstacle, we developed a VCP inhibitor substrate trapping approach (VISTA) to identify endogenous ERAD substrates. VISTA exploits the small-molecule VCP inhibitor CB5083 to trap ERAD substrates in a membrane-associated, ubiquitinated form. This strategy, coupled with quantitative ubiquitin proteomics, identified previously validated (e.g., ApoB100, Insig2, and DHCR7) and novel (e.g., SCD1 and RNF5) ERAD substrates in cultured human hepatocellular carcinoma cells. Moreover, our results indicate that RNF5 autoubiquitination on multiple lysine residues targets it for ubiquitin and VCP-­dependent clearance. Thus, VISTA provides a generalizable discovery method that expands the available toolbox of strategies to elucidate the ERAD substrate landscape.

scholarly journals The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades  doublecortin proteins to constrain neuronal dendritogenesis

2020 ◽  
pp. jbc.RA120.016210
Author(s):  
Jianing Song ◽  
Ronald A. Merrill ◽  
Andrew Y. Usachev ◽  
Stefan Strack
Keyword(s):  
Intellectual Disability ◽  
Brain Development ◽  
Ubiquitin Ligase ◽  
Hippocampal Neurons ◽  
E3 Ubiquitin Ligase ◽  
Gene Mutations ◽  
Disease Protein ◽  
Associated Proteins ◽  
Interaction Regions ◽  
And Function

Proper brain development and function requires finely controlled mechanisms for protein turnover and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3 (Cul3)-containing E3 ubiquitin ligases and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability (XLID). Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 (DCLK1/2) as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.

scholarly journals NOD1 is super-activated through spatially-selective ubiquitination by the Salmonella effector SspH2

2021 ◽  
Author(s):  
Cole Delyea ◽  
Shu Luo ◽  
Bradley E Dubrule ◽  
Olivier Julien ◽  
Amit P Bhavsar
Keyword(s):  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Inflammatory Cytokine ◽  
E3 Ubiquitin Ligase ◽  
Cytokine Secretion ◽  
Host Cells ◽  
List Type ◽  
The Novel ◽  
Inflammatory Signaling ◽  
Lysine Residues

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the novel E3 ubiquitin ligase family, interacts with, and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report the novel discovery that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling that results from targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture. We also show that SspH2 specifically interacts with the NBD and LRR domains of NOD1 and super-activates NOD1- and NOD2-mediated cytokine secretion via the NF-κB pathway. Mass spectrometry analyses identified lysine residues in NOD1 that were ubiquitinated after interaction with SspH2. Through NOD1 mutational analyses, we identified four key lysine residues that are required for NOD1 super-activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on NOD1 that is targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin, and uncovers a unique mechanism of spatially-selective ubiquitination to enhance the activation of an archetypal NLR.

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