Oligomerization of Selective Autophagy Receptors for the Targeting and Degradation of Protein Aggregates

The selective targeting and disposal of solid protein aggregates are essential for cells to maintain protein homoeostasis. Autophagy receptors including p62, NBR1, Cue5/TOLLIP (CUET), and Tax1-binding protein 1 (TAX1BP1) proteins function in selective autophagy by targeting ubiquitinated aggregates through ubiquitin-binding domains. Here, we summarize previous beliefs and recent findings on selective receptors in aggregate autophagy. Since there are many reviews on selective autophagy receptors, we focus on their oligomerization, which enables receptors to function as pathway determinants and promotes phase separation.

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Aberrant Stress Granule Dynamics and Aggrephagy in ALS Pathogenesis

Cells ◽

10.3390/cells10092247 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2247

Author(s):

Yi Zhang ◽

Jiayu Gu ◽

Qiming Sun

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Phase Separation ◽

Gene Mutations ◽

Stress Granules ◽

Protein Aggregates ◽

Toxic Protein ◽

Selective Autophagy ◽

Dynamic Assembly ◽

Autophagy Pathway ◽

Lateral Sclerosis

Stress granules are conserved cytosolic ribonucleoprotein (RNP) compartments that undergo dynamic assembly and disassembly by phase separation in response to stressful conditions. Gene mutations may lead to aberrant phase separation of stress granules eliciting irreversible protein aggregations. A selective autophagy pathway called aggrephagy may partially alleviate the cytotoxicity mediated by these protein aggregates. Cells must perceive when and where the stress granules are transformed into toxic protein aggregates to initiate autophagosomal engulfment for subsequent autolysosomal degradation, therefore, maintaining cellular homeostasis. Indeed, defective aggrephagy has been causally linked to various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In this review, we discuss stress granules at the intersection of autophagy and ALS pathogenesis.

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The Ubiquitin versus NEDD8 Binding Preference of NEDD4 Binding Protein 1 (N4BP1) Is Based on a Mutual Conformational Perturbation

10.20944/preprints201902.0213.v1 ◽

2019 ◽

Author(s):

Ridvan Nepravishta ◽

Federica Ferrentino ◽

Walter Mandaliti ◽

Anna Mattioni ◽

Luisa Castagnoli ◽

...

Keyword(s):

Molecular Mechanisms ◽

Binding Protein ◽

Functional Characterization ◽

Binding Domain ◽

Functional Domain ◽

Protein Sequence Analysis ◽

Domain Family ◽

Hydrophobic Patch ◽

Binding Domains ◽

Ubiquitin Binding

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44 (Leu-8, Ile-44, Val-70). Nevertheless, a variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD recognizes ubiquitin. Here, we describe the structure of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis and NMR spectroscopy of the 15N isotopically labelled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the CUE (Coupling of ubiquitin conjugation to ER degradation) domain family, where an invariant proline, usually following a phenylalanine, is required for binding to ubiquitin. Interestingly, the UBD of N4BP1 is evolutionary related to CUBAN (Cullin binding domain associating with NEDD8) (40% identity and 47% similarity), a protein module that also recognizes the ubiquitin-like NEDD8, which is the closest relative of ubiquitin (58% identity and 80% similarity). By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the UBD of N4BP1 lacks the NEDD8 binding properties observed in CUBAN and it recognizes the Ile44-centered patch of ubiquitin through a dedicated binding site, which share some of the features observed in the CUE domain family. Moreover, we show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both the CUBAN and the UBD of N4BP1 are poly-ubiquitinated in cells. This modification is dependent on the presence of a functional domain. We believe that the structural and functional characterization of this novel UBD will allow a deeper understanding of the molecular mechanisms governing N4BP1 function, while at the same time providing a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

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Selective Autophagy by Close Encounters of the Ubiquitin Kind

Cells ◽

10.3390/cells9112349 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2349 ◽

Cited By ~ 1

Author(s):

Anna Vainshtein ◽

Paolo Grumati

Keyword(s):

Protein Aggregates ◽

Degradation Process ◽

Eukaryotic Cells ◽

Post Translational Modifications ◽

Selective Autophagy ◽

Close Encounters ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain ◽

Cellular Turnover

Autophagy, a bulk degradation process within eukaryotic cells, is responsible for cellular turnover and nutrient liberation during starvation. Increasing evidence indicate that this process can be extremely discerning. Selective autophagy segregates and eliminates protein aggregates, damaged organelles, and invading organisms. The specificity of this process is largely mediated by post-translational modifications (PTMs), which are recognized by autophagy receptors. These receptors grant autophagy surgical precision in cargo selection, where only tagged substrates are engulfed within autophagosomes and delivered to the lysosome for proteolytic breakdown. A growing number of selective autophagy receptors have emerged including p62, NBR1, OPTN, NDP52, TAX1BP1, TOLLIP, and more continue to be uncovered. The most well-documented PTM is ubiquitination and selective autophagy receptors are equipped with a ubiquitin binding domain and an LC3 interacting region which allows them to physically bridge cargo to autophagosomes. Here, we review the role of ubiquitin and ubiquitin-like post-translational modifications in various types of selective autophagy.

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Autophagy Receptor Protein Tax1-Binding Protein 1/TRAF6-Binding Protein Is a Cellular Substrate of Enteroviral Proteinase

Frontiers in Microbiology ◽

10.3389/fmicb.2021.647410 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yasir Mohamud ◽

Yuan Chao Xue ◽

Huitao Liu ◽

Chen Seng Ng ◽

Amirhossein Bahreyni ◽

...

Keyword(s):

Binding Protein ◽

Receptor Protein ◽

Late Infection ◽

Culture Studies ◽

Selective Autophagy ◽

Protein Levels ◽

Cvb3 Infection ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain ◽

Autophagy Pathway

Enteroviruses (EVs) usurp the host autophagy pathway for pro-viral functions; however, the consequence of EV-induced diversion of autophagy on organelle quality control is poorly defined. Using coxsackievirus B3 (CVB3) as a model EV, we explored the interplay between EV infection and selective autophagy receptors, i.e., Tax1-binding protein 1/TRAF6-binding protein (T6BP), optineurin (OPTN), and nuclear dot 10 protein 52 (NDP52), known to be involved in regulating the clearance of damaged mitochondria, a process termed as mitophagy. Following CVB3 infection, we showed significant perturbations of the mitochondrial network coincident with degradation of the autophagy receptor protein T6BP, similar phenomenon to what we previously observed on NDP52. Notably, protein levels of OPTN are not altered during early infection and slightly reduced upon late infection. Cell culture studies revealed that T6BP degradation occurs independent of the function of host caspases and viral proteinase 3C, but requires the proteolytic activity of viral proteinase 2A. Further investigation identified the cleavage site on T6BP after the amino acid 621 that separates the C-terminal ubiquitin-binding domain from the other functional domains at the N-terminus. Genetic silencing of T6BP and OPTN results in the attenuation of CVB3 replication, suggesting a pro-viral activity for these two proteins. Finally, functional assessment of cleaved fragments from NDP52 and T6BP revealed abnormal binding affinity and impaired capacity to be recruited to depolarized mitochondria. Collectively, these results suggest that CVB3 targets autophagy receptors to impair selective autophagy.

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Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

The Journal of Cell Biology ◽

10.1083/jcb.200711108 ◽

2008 ◽

Vol 180 (6) ◽

pp. 1065-1071 ◽

Cited By ~ 219

Author(s):

Ioannis P. Nezis ◽

Anne Simonsen ◽

Antonia P. Sagona ◽

Kim Finley ◽

Sébastien Gaumer ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Protein Aggregates ◽

Adult Brain ◽

Aggregate Formation ◽

Ubiquitinated Protein ◽

Physiological Conditions ◽

Binding Domains ◽

Ubiquitin Binding ◽

Autophagic Degradation

p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

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146: Clusterin is a Ubiquitin Binding Protein Involved in Murr1 and 1-KB Proteasomal Degradation to Confer Cell Survival in Prostate Cancer

The Journal of Urology ◽

10.1016/s0022-5347(18)30411-7 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 50-50

Author(s):

Amina Zoubeidi ◽

Eliana Beraldi ◽

Anousheh Zardan ◽

Martin E. Gleave

Keyword(s):

Prostate Cancer ◽

Cell Survival ◽

Binding Protein ◽

Proteasomal Degradation ◽

Ubiquitin Binding

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Faculty Opinions recommendation of Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738178126.793576688 ◽

2020 ◽

Author(s):

Athi N Naganathan

Keyword(s):

Phase Separation ◽

Nucleic Acid ◽

Binding Protein ◽

Nucleic Acid Binding ◽

Nucleic Acid Binding Protein ◽

Acid Binding Protein

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Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

Science Advances ◽

10.1126/sciadv.abg4922 ◽

2021 ◽

Vol 7 (17) ◽

pp. eabg4922

Author(s):

Chunmei Chang ◽

Xiaoshan Shi ◽

Liv E. Jensen ◽

Adam L. Yokom ◽

Dorotea Fracchiolla ◽

...

Keyword(s):

Complex I ◽

Kinase Activity ◽

Protein Aggregates ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Giant Unilamellar Vesicles ◽

Core Complex ◽

Selective Autophagy ◽

The Core ◽

Stimulation Of

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

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