Proteaphagy in Mammalian Cells Can Function Independent of ATG5/ATG7

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), β-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Because these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.

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The p97-UBXN1 complex regulates aggresome formation

10.1101/2020.09.03.281766 ◽

2020 ◽

Author(s):

Sirisha Mukkavalli ◽

Jacob Aaron Klickstein ◽

Betty Ortiz ◽

Peter Juo ◽

Malavika Raman

Keyword(s):

Protein Aggregation ◽

Mammalian Cells ◽

Proteasome Inhibition ◽

Adaptor Proteins ◽

Ubiquitin Proteasome System ◽

Aaa Atpase ◽

Evolutionarily Conserved ◽

Ubiquitin Proteasome ◽

Aggresome Formation

AbstractThe recognition and disposal of misfolded proteins are essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. It is presently unclear how diverse disease-relevant aggregates are recognized and processed for degradation. The p97 AAA-ATPase in combination with a host of adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the ubiquitin-proteasome system or through autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear given the large number of pathways that rely on p97 function. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes which are membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates both aggresome formation and clearance in proteasome-inhibited cells. We identify a novel and specific role for the p97 adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to ubiquitin-positive aggresomes and UBXN1 knockout cells are unable to form a single aggresome, and instead display dispersed ubiquitin aggregates. Furthermore, loss of p97-UBXN1 results in the increase in Huntingtin polyQ aggregates both in mammalian cells as well as in a C.elegans model of Huntington’s Disease. Together our work identifies evolutionarily conserved roles for p97 and its adaptor UBXN1 in the disposal of protein aggregates.

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Noncoding RNA MaIL1 is an integral component of the TLR4–TRIF pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920393117 ◽

2020 ◽

Vol 117 (16) ◽

pp. 9042-9053 ◽

Cited By ~ 4

Author(s):

Marina Aznaourova ◽

Harshavardhan Janga ◽

Stephanie Sefried ◽

Andreas Kaufmann ◽

Jens Dorna ◽

...

Keyword(s):

Mammalian Cells ◽

Noncoding Rna ◽

Signal Transduction Pathway ◽

Ubiquitin Proteasome System ◽

Lavage Fluid ◽

Cellular Protein ◽

Type I ◽

Toll Like Receptor 4 ◽

Ubiquitin Proteasome ◽

Highly Correlated

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin–proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification–mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4–TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

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Ochratoxin A Sequentially Activates Autophagy and the Ubiquitin-Proteasome System

Toxins ◽

10.3390/toxins11110615 ◽

2019 ◽

Vol 11 (11) ◽

pp. 615

Author(s):

Akpinar ◽

Kahraman ◽

Yaman

Keyword(s):

Ochratoxin A ◽

Early Time ◽

Human Kidney ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Mouse Embryonic Fibroblast ◽

Proteasomal Activity ◽

Ubiquitin Proteasome ◽

Autophagic Activity ◽

Sustained Activation

Ochratoxin A (OTA) is a carcinogenic mycotoxin, which is produced by Aspergillus and Penicillium genera of fungi and commonly contaminates food and feed. We and others have previously shown that OTA causes sustained activation of PI3K/AKT and MAPK/ERK1-2 signaling pathways in different cell types and animal models. Given the close relationship between cellular signaling activity and protein stability, we were curious whether increased PI3K/AKT and MAPK/ERK1-2 signaling may be the result of OTA-stimulated alterations in proteolytic activity. We show that both of the major proteolytic systems, autophagy, and the ubiquitin-proteasome system (UPS), are activated upon OTA exposure in human kidney proximal tubule HK-2 and mouse embryonic fibroblast (MEF) cells. OTA stimulates transient autophagic activity at early time points of treatment but autophagic activity subsides after 6 h even in the sustained presence of OTA. Interestingly, OTA exposure also results in increased cell death in wild-type MEF cells but not in autophagy-halted Atg5-deficient cells, suggesting that autophagy exerts a pro-death effect on OTA-induced cytotoxicity. In addition, prolonged OTA exposure decreased ubiquitinated protein levels by increasing proteasomal activity. Using purified and cellular proteasomes, we observed enhanced chymotrypsin-, caspase-, and trypsin-like activities of the 26S but not the 20S proteasome in the presence of OTA. However, in the cellular context, increased proteasomal activity depended on prior induction of autophagy. Our results suggest that autophagy and subsequent UPS activation are responsible for sustained activation of PI3K/AKT and MAPK/ERK1-2 pathways through regulating the levels of critical phosphatases VHR/DUSP3, DUSP4, and PHLPP, which are known to be involved in OTA toxicity and carcinogenicity.

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Use of Ubiquitin-Proteasome System Profiling for Differentiating Between Various Leukemic Processes.

Blood ◽

10.1182/blood.v114.22.4712.4712 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4712-4712

Author(s):

Ke Zhang ◽

Hagop M. Kantarjian ◽

Wanlong Ma ◽

XI Zhang ◽

Xiuqiang Wang ◽

...

Keyword(s):

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Direct Analysis ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Lymphocytic Leukemia ◽

Ubiquitin Proteasome

Abstract Abstract 4712 The ubiquitin-proteasome system (UPS) plays a major role in cell homeostasis in normal and neoplastic states. Expression and function of the UPS system vary with the specific characteristics of individual cell types, suggesting that determination of UPS “signatures” could be useful in identifying various cell populations. Since direct analysis of cancer cells is often problematic, even in hematologic diseases, we explored the potential of using UPS signatures in plasma to differentiate between various leukemias. We first analyzed plasma UPS profiles of patients with acute myeloid leukemia (AML; n=111), acute lymphoblastic leukemia (ALL; n=29), advanced myelodysplastic syndrome (MDS; n=20), chronic lymphocytic leukemia (CLL; n=118), or chronic myeloid leukemia (CML; n=128; 46 in accelerated/blast crisis [ACC/BL], 82 in chronic phase), and 85 healthy control subjects. Plasma levels of proteasome, ubiquitin (poly-ubiquitin), and the 3 proteasome enzymatic activities (chymotrypsin-like [Ch-L], caspase-like [Cas-L], trypsin-like [Tr-L]) were measured. Specific activities were calculated by normalizing each of the 3 enzyme activities to the levels of proteasome protein in plasma (Ch-L/p, Cas-L/p, and Tr-L/p). These 8 variables were used in multivariate logistic regression models to differentiate between leukemic processes. UPS signatures provided clear differentiation between patients with a leukemic process and normal controls (AUC=0.991), using 6 different variables (Tr-L/P, Ch-L, Ch-L/p, Cas-L, Cas-L/P, ubiquitin). Distinguishing between acute (AML, ALL, MDS) and chronic (CML, CLL) processes was less efficient (AUC=0.853 using Tr-L, Tr-L/P, Cas-L/P, Ch-L/P, proteasome, Ch-L), likely due to the high proportion (36%) of CML patients in ACC/BL phase. However, UPS signatures generally yielded powerful differentiation between individual leukemias (Table). MDS was not well differentiated from AML (AUC=0.791), reflecting the significant biological overlap of these diseases. These data support the potential usefulness of the UPS profile to aid in the differential diagnosis of various leukemias. Disclosures: No relevant conflicts of interest to declare.

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EVI/WLS function is regulated by ubiquitination and linked to ER-associated degradation by ERLIN2

Journal of Cell Science ◽

10.1242/jcs.257790 ◽

2021 ◽

Author(s):

Lucie M. Wolf ◽

Annika M. Lambert ◽

Julie Haenlin ◽

Michael Boutros

Keyword(s):

Mammalian Cells ◽

Secretory Pathway ◽

Ubiquitin Proteasome System ◽

E3 Ligases ◽

Wnt Proteins ◽

Interaction Partners ◽

Ubiquitin Proteasome ◽

Endogenous Substrate ◽

Ubiquitin Conjugating Enzymes ◽

Ubiquitination Machinery

WNT signalling is important for development in all metazoan animals and is associated with various human diseases. The Ubiquitin-Proteasome System (UPS) and regulatory ER-associated degradation (ERAD) have been implicated in the production of WNT proteins. Here, we investigated how the WNT secretory factor EVI/WLS is ubiquitinated, recognised by ERAD components and subsequently removed from the secretory pathway. We performed a focused, immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitination machinery. Interestingly, we found in addition that EVI/WLS is ubiquitinated and degraded in cells irrespective of their level of WNT production. This K11, K48, and K63-linked ubiquitination is mediated by the E2 ubiquitin conjugating enzymes UBE2J2, UBE2K, and UBE2N, but independent of the E3 ligases HRD1/SYVN or GP78/AMFR. Taken together, our study identified factors that link the UPS to the WNT secretory pathway and provides mechanistic details on the fate of an endogenous substrate of regulatory ERAD in mammalian cells.

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Co-Transmission of Alpha-Synuclein and TPPP/p25 Inhibits Their Proteolytic Degradation in Human Cell Models

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.666026 ◽

2021 ◽

Vol 8 ◽

Author(s):

Attila Lehotzky ◽

Judit Oláh ◽

János Tibor Fekete ◽

Tibor Szénási ◽

Edit Szabó ◽

...

Keyword(s):

Cell Types ◽

Ubiquitin Proteasome System ◽

Alpha Synuclein ◽

Human Cells ◽

Proteolytic Degradation ◽

Intrinsically Disordered ◽

Cell Transmission ◽

Key Factor ◽

Ubiquitin Proteasome ◽

Combined Strategy

The pathological association of alpha-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25) is a key factor in the etiology of synucleinopathies. In normal brains, the intrinsically disordered SYN and TPPP/p25 are not found together but exist separately in neurons and oligodendrocytes, respectively; in pathological states, however, they are found in both cell types due to their cell-to-cell transmission. The autophagy degradation of the accumulated/assembled SYN has been considered as a potential therapeutic target. We have shown that the hetero-association of SYN with TPPP/p25 after their uptake from the medium by human cells (which mimics cell-to-cell transmission) inhibits both their autophagy- and the ubiquitin-proteasome system-derived elimination. These results were obtained by ELISA, Western blot, FACS and immunofluorescence confocal microscopy using human recombinant proteins and living human cells; ANOVA statistical analysis confirmed that TPPP/p25 counteracts SYN degradation by hindering the autophagy maturation at the stage of LC3B-SQSTM1/p62-derived autophagosome formation and its fusion with lysosome. Recently, fragments of TPPP/p25 that bind to the interface between the two hallmark proteins have been shown to inhibit their pathological assembly. In this work, we show that the proteolytic degradation of SYN on its own is more effective than when it is complexed with TPPP/p25. The combined strategy of TPPP/p25 fragments and proteolysis may ensure prevention and/or elimination of pathological SYN assemblies.

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Bifunctional Small Molecules That Mediate the Degradation of Extracellular Proteins

10.26434/chemrxiv.12732689.v1 ◽

2020 ◽

Author(s):

David Caianiello ◽

Mengwen Zhang ◽

Jason Ray ◽

Jake Swartzel ◽

Emily Branham ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Extracellular Proteins ◽

Target Protein ◽

Disease Treatment ◽

Bifunctional Molecules ◽

Synthetic Molecules ◽

Ubiquitin Proteasome ◽

Mode A

Targeted protein degradation (TPD) has emerged as a promising and exciting therapeutic strategy. The majority of existing TPD technologies rely on the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modularly designed, bifunctional synthetic molecules called MoDE-As (Molecular Degraders of Extracellular proteins through the Asialoglycoprotein receptor (ASGPR)), which are capable of mediating the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and the ASGPR, which is expressed primarily on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing bifunctional molecules that induce the degradation of both antibody and pro-inflammatory cytokine proteins. To our knowledge, these data represent the first experimental evidence that non-proteinogenic, synthetic molecules can be employed for the TPD of extracellular proteins both in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

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Role of the COP9 Signalosome (CSN) in Cardiovascular Diseases

Biomolecules ◽

10.3390/biom9060217 ◽

2019 ◽

Vol 9 (6) ◽

pp. 217 ◽

Cited By ~ 6

Author(s):

Milic ◽

Tian ◽

Bernhagen

Keyword(s):

Cardiovascular Diseases ◽

Molecular Mechanisms ◽

Three Dimensional ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Cop9 Signalosome ◽

Ring E3 Ligase ◽

Ubiquitin Proteasome ◽

Underlying Mechanisms ◽

Isopeptidase Activity

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is an evolutionarily conserved multi-protein complex, consisting of eight subunits termed CSN1-CSN8. The main biochemical function of the CSN is the control of protein degradation via the ubiquitin-proteasome-system through regulation of cullin-RING E3-ligase (CRL) activity by deNEDDylation of cullins, but the CSN also serves as a docking platform for signaling proteins. The catalytic deNEDDylase (isopeptidase) activity of the complex is executed by CSN5, but only efficiently occurs in the three-dimensional architectural context of the complex. Due to its positioning in a central cellular pathway connected to cell responses such as cell-cycle, proliferation, and signaling, the CSN has been implicated in several human diseases, with most evidence available for a role in cancer. However, emerging evidence also suggests that the CSN is involved in inflammation and cardiovascular diseases. This is both due to its role in controlling CRLs, regulating components of key inflammatory pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and complex-independent interactions of subunits such as CSN5 with inflammatory proteins. In this case, we summarize and discuss studies suggesting that the CSN may have a key role in cardiovascular diseases such as atherosclerosis and heart failure. We discuss the implicated molecular mechanisms ranging from inflammatory NF-κB signaling to proteotoxicity and necrosis, covering disease-relevant cell types such as myeloid and endothelial cells or cardiomyocytes. While the CSN is considered to be disease-exacerbating in most cancer entities, the cardiovascular studies suggest potent protective activities in the vasculature and heart. The underlying mechanisms and potential therapeutic avenues will be critically discussed.

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A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0965 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1953-1963 ◽

Cited By ~ 36

Author(s):

Nazli Ghaboosi ◽

Raymond J. Deshaies

Keyword(s):

Initial Step ◽

Adaptor Proteins ◽

Ubiquitin Proteasome System ◽

Temperature Sensitive ◽

Polyubiquitin Chains ◽

Multiple Substrates ◽

Ubiquitin Proteasome ◽

Sensitive Allele

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.

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