ubiquitin binding
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2022 ◽  
Author(s):  
Megha Bansal ◽  
Kapil Sirohi ◽  
Shivranjani C Moharir ◽  
Ghanshyam Swarup

Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/-MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.


2022 ◽  
Author(s):  
Fei Huang ◽  
Yu Hui ◽  
Ang Li ◽  
Rishalaiti Tayier ◽  
Dilinaer Yaermaimaiti ◽  
...  

Abstract Endemic arsenism is a major disease concern in China, with arsenic poisoning and induced potential lesions key issues on a global level. The liver is the main target organ where arsenic is metabolized; chronic exposure to arsenic-induced liver fibrosis is also closely related to autophagy, however, the exact mechanisms are remain unclear. In this study, we explored the effects of NaAsO2 on apoptosis and autophagy in human hepatic stellate cells(HSC). We established a fibrosis model in the HSC line, LX-2 which was exposed to NaAsO2 for 24h, 48h, and 72h. Cells were then transfected using an autophagy double-labeled RFP-GFP-LC3 adenoviral plasmid. Laser confocal microscopy indicated significant infection efficiencies and autophagy in LX-2. Flow cytometry was also used to investigate the effects of different NaAsO2 doses on apoptosis. NaAsO2 treatment upregulated the expression of autophagic markers, including microtubule-associated protein light chain A/B(LC3), ubiquitin binding protein(SQSTM-1/P62), autophagy related genes(ATGs), recombinant human autophagy effector protein (Beclin-1), and B cell lymphoma-2(BCL-2), but downregulated mammalian target of rapamycin(mTOR). Also, α-smooth muscle actin(α-SMA) expression was significantly upregulated in all NaAsO2 groups. Furthermore, mTOR silencing via 3-methyladenine(3-MA) altered NaAsO2 induced autophagy, LC3, Beclin-1, and SQSTM-1/P62 expression were all upregulated in both NaAsO2 and 3-MA-iAs groups. Altogether, NaAsO2 induced HSC autophagy via apoptotic pathways. 3-MA inhibited LX-2 activity and reduced NaAsO2-induced autophagy which may inhibit fibrosis progression caused by this toxin.


Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 215
Author(s):  
Ravinder Kumar ◽  
Ankit Shroff ◽  
Taras Y. Nazarko

Recently, we developed Komagataella phaffii (formerly Pichia pastoris) as a model for lipophagy, the selective autophagy of lipid droplets (LDs). We found that lipophagy pathways induced by acute nitrogen (N) starvation and in stationary (S) phase have different molecular mechanisms. Moreover, both types of lipophagy are independent of Atg11, the scaffold protein that interacts with most autophagic receptors and, therefore, is essential for most types of selective autophagy in yeast. Since yeast aggrephagy, the selective autophagy of ubiquitinated protein aggregates, is also independent of Atg11 and utilizes the ubiquitin-binding receptor, Cue5, we studied the relationship of K. phaffii Cue5 with differentially induced LDs and lipophagy. While there was no relationship of Cue5 with LDs and lipophagy under N-starvation conditions, Cue5 accumulated on LDs in S-phase and degraded together with LDs via S-phase lipophagy. The accumulation of Cue5 on LDs and its degradation by S-phase lipophagy strongly depended on the ubiquitin-binding CUE domain and Prl1, the positive regulator of lipophagy 1. However, unlike Prl1, which is required for S-phase lipophagy, Cue5 was dispensable for it suggesting that Cue5 is rather a new substrate of this pathway. We propose that a similar mechanism (Prl1-dependent accumulation on LDs) might be employed by Prl1 to recruit another ubiquitin-binding protein that is essential for S-phase lipophagy.


Author(s):  
Ravinder Kumar ◽  
Ankit Shroff ◽  
Taras Y. Nazarko

Recently, we developed Komagataella phaffii (formerly Pichia pastoris) as a model for lipophagy, the selective autophagy of lipid droplets (LDs). We found that lipophagy pathways induced by acute nitrogen (N) starvation and in stationary (S) phase have different molecular mechanisms. Moreover, both types of lipophagy are independent of Atg11, the scaffold protein that interacts with most autophagic receptors and, therefore, is essential for most types of selective autophagy in yeast. Since yeast aggrephagy, the selective autophagy of ubiquitinated protein aggregates, is also independent of Atg11 and utilizes the ubiquitin-binding receptor, Cue5, we studied the relationship of K. phaffii Cue5 with differentially induced LDs and lipophagy. While there was no relationship of Cue5 with LDs and lipophagy under N-starvation conditions, Cue5 accumulated on LDs in S-phase and degraded together with LDs via S-phase lipophagy. The accumulation of Cue5 on LDs and its degradation by S-phase lipophagy strongly depended on the ubiquitin-binding CUE domain and Prl1, the positive regulator of lipophagy 1. However, unlike Prl1, which is required for S-phase lipophagy, Cue5 was dispensable for it suggesting that Cue5 is rather a new substrate of this pathway. We propose that a similar mechanism (Prl1-dependent accumulation on LDs) might be employed by Prl1 to recruit another ubiquitin-binding protein that is essential for S-phase lipophagy.


Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6280
Author(s):  
Eugenia Passaro ◽  
Chiara Papulino ◽  
Ugo Chianese ◽  
Antonella Toraldo ◽  
Raffaella Congi ◽  
...  

Autophagy is an essential intracellular catabolic mechanism involved in the degradation and recycling of damaged organelles regulating cellular homeostasis and energy metabolism. Its activation enhances cellular tolerance to various stresses and is known to be involved in drug resistance. In cancer, autophagy has a dual role in either promoting or blocking tumorigenesis, and recent studies indicate that epigenetic regulation is involved in its mechanism of action in this context. Specifically, the ubiquitin-binding histone deacetylase (HDAC) enzyme HDAC6 is known to be an important player in modulating autophagy. Epigenetic modulators, such as HDAC inhibitors, mediate this process in different ways and are already undergoing clinical trials. In this review, we describe current knowledge on the role of epigenetic modifications, particularly HDAC-mediated modifications, in controlling autophagy in cancer. We focus on the controversy surrounding their ability to promote or block tumor progression and explore the impact of HDAC6 inhibitors on autophagy modulation in cancer. In light of the fact that targeted drug therapy for cancer patients is attracting ever increasing interest within the research community and in society at large, we discuss the possibility of using HDAC6 inhibitors as adjuvants and/or in combination with conventional treatments to overcome autophagy-related mechanisms of resistance.


2021 ◽  
Author(s):  
Mengwen Zhang ◽  
Jason M. Berk ◽  
Adrian B. Mehrtash ◽  
Jean Kanyo ◽  
Mark Hochstrasser

AbstractProtein ubiquitylation is an important post-translational modification affecting an wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on developing methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain derived from an Orientia tsutsugamushi deubiquitylase. We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As a proof-of-principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin researchers with specific advantages over other methods, such as in detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.


2021 ◽  
Author(s):  
Swapneeta S Date ◽  
Peng Xu ◽  
Nathaniel L. Hepowit ◽  
Nicholas S. Diab ◽  
Jordan Best ◽  
...  

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime COPI assembly to ensure incorporation of these essential cargoes into vesicles but the regulation of these events is poorly understood. Here we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs. The ability of COPI to bind ubiquitin through its N-terminal WD repeat domain of β′COP or through an unrelated ubiquitin-binding domain (UBD) is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is thought to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.


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