scholarly journals Protein import into chloroplasts and its regulation by the ubiquitin-proteasome system

2020 ◽  
Vol 48 (1) ◽  
pp. 71-82 ◽  
Author(s):  
Simon M. Thomson ◽  
Pablo Pulido ◽  
R. Paul Jarvis
Keyword(s):  
Protein Delivery ◽  
Protein Import ◽  
Ubiquitin Proteasome System ◽  
Dependent Manner ◽  
Environmental Signals ◽  
Selective Degradation ◽  
Precursor Proteins ◽  
Ubiquitin Proteasome ◽  
Energy Dependent ◽  
Envelope Membranes

Chloroplasts are photosynthetic plant organelles descended from a bacterial ancestor. The vast majority of chloroplast proteins are synthesized in the cytosol and then imported into the chloroplast post-translationally. Translocation complexes exist in the organelle's outer and inner envelope membranes (termed TOC and TIC, respectively) to facilitate protein import. These systems recognize chloroplast precursor proteins and mediate their import in an energy-dependent manner. However, many unanswered questions remain regarding mechanistic details of the import process and the participation and functions of individual components; for example, the cytosolic events that mediate protein delivery to chloroplasts, the composition of the TIC apparatus, and the nature of the protein import motor all require resolution. The flux of proteins through TOC and TIC varies greatly throughout development and in response to specific environmental cues. The import process is, therefore, tightly regulated, and it has emerged that the ubiquitin-proteasome system (UPS) plays a key role in this regard, acting at several different steps in the process. The UPS is involved in: the selective degradation of transcription factors that co-ordinate the expression of chloroplast precursor proteins; the removal of unimported chloroplast precursor proteins in the cytosol; the inhibition of chloroplast biogenesis pre-germination; and the reconfiguration of the TOC apparatus in response to developmental and environmental signals in a process termed chloroplast-associated protein degradation. In this review, we highlight recent advances in our understanding of protein import into chloroplasts and how this process is regulated by the UPS.

scholarly journals Identification of Protein Transport Complexes in the Chloroplastic Envelope Membranes via Chemical Cross-Linking

1997 ◽  
Vol 136 (5) ◽  
pp. 983-994 ◽  
Author(s):  
Mitsuru Akita ◽  
Erik Nielsen ◽  
Kenneth Keegstra
Keyword(s):  
Membrane Proteins ◽  
Protein Transport ◽  
Protein Import ◽  
Cross Linking ◽  
Envelope Membrane ◽  
Precursor Proteins ◽  
Intact Chloroplasts ◽  
Outer Envelope Membrane ◽  
Envelope Membranes ◽  
Chemical Cross Linking

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.

scholarly journals Macrolide Antibiotics Block Autophagy Flux and Sensitize to Bortezomib Via Endoplasmic Reticulum-Stress-Mediated CHOP Induction in Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4992-4992
Author(s):  
Shota Moriya ◽  
Xiao-Fang Che ◽  
Seiichiro Komatsu ◽  
Akihisa Abe ◽  
Tomohiro Kawaguchi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Lines ◽  
Macrolide Antibiotic ◽  
Combined Treatment ◽  
Ubiquitin Proteasome System ◽  
Macrolide Antibiotics ◽  
Autophagy Flux ◽  
Selective Degradation ◽  
Ubiquitin Proteasome

Abstract Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved cellular process of self-degradation in eukaryotes. Intracellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intracellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) cell lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM cell lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins, and induced the proapoptotic transcription factor CHOP. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. BIM, BAX, DR5, and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and up-regulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER-stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sensitivity of Acute Myelocytic Leukemia Cells to the Dienone Compound VLX1570 Is Associated with Inhibition of the Ubiquitin-Proteasome System

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1339
Author(s):  
Karthik Selvaraju ◽  
Kourosh Lotfi ◽  
Johannes Gubat ◽  
Maria Miquel ◽  
Amanda Nilsson ◽  
...  
Keyword(s):  
Cell Viability ◽  
Drug Exposure ◽  
Cancer Therapeutics ◽  
Ubiquitin Proteasome System ◽  
Glutathione Depletion ◽  
Acute Myelocytic Leukemia ◽  
Dependent Manner ◽  
Turnover Rates ◽  
Ubiquitin Proteasome

Dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been widely reported to show tumor cell selectivity. These compounds target the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. The induction of oxidative stress, depletion of glutathione, and induction of high-molecular-weight (HMW) complexes have also been reported. We here examined the response of acute myeloid leukemia (AML) cells to the dienone compound VLX1570. AML cells have relatively high protein turnover rates and have also been reported to be sensitive to depletion of reduced glutathione. We found AML cells of diverse cytogenetic backgrounds to be sensitive to VLX1570, with drug exposure resulting in an accumulation of ubiquitin complexes, induction of ER stress, and the loss of cell viability in a dose-dependent manner. Caspase activation was observed but was not required for the loss of cell viability. Glutathione depletion was also observed but did not correlate to VLX1570 sensitivity. Formation of HMW complexes occurred at higher concentrations of VLX1570 than those required for the loss of cell viability and was not enhanced by glutathione depletion. To study the effect of VLX1570 we developed a zebrafish PDX model of AML and confirmed antigrowth activity in vivo. Our results show that VLX1570 induces UPS inhibition in AML cells and encourage further work in developing compounds useful for cancer therapeutics.

scholarly journals Exercise Training-Increased FBXO32 and FOXO1 in a Gender-Dependent Manner in Mild Cognitively Impaired African Americans: GEMS-1 Study

2021 ◽  
Vol 13 ◽  
Author(s):  
Fikru B. Bedada ◽  
Oyonumo E. Ntekim ◽  
Evaristus O. Nwulia ◽  
Thomas V. Fungwe ◽  
Sheeba Raaj Nadarajah ◽  
...  
Keyword(s):  
African Americans ◽  
Gene Expression Analysis ◽  
Ubiquitin Proteasome System ◽  
Cognitively Impaired ◽  
Dependent Manner ◽  
Specific Exercise ◽  
Ubiquitin Proteasome ◽  
The Brain ◽  
Gender Specific

The ubiquitin proteasome system (UPS) and FOXOs transcription factors play a pivotal role in cellular clearance and minimizing the accumulation of Aβ in neurodegeneration (ND). In African Americans (AAs) with mild cognitive impairment (MCI), the role of components of UPS and FOXOs; and whether they are amenable to exercise effects is unknown. We hypothesized that exercise can enhance cellular clearance systems during aging and ND by increasing expressions of FBXO32 and FOXO1. To test this hypothesis, we used TaqMan gene expression analysis in peripheral blood (PB) to investigate the component of UPS and FOXOs; and provide mechanistic insight at baseline, during exercise, and in both genders. At baseline, levels of FBXO32 were higher in women than in men. In our attempt to discern gender-specific exercise-related changes, we observed that levels of FBXO32 increased in men but not in women. Similarly, levels of FOXO1 increased in men only. These data suggest that a graded dose of FBXO32 and FOXO1 may be beneficial when PB cells carrying FBXO32 and FOXO1 summon into the brain in response to Alzheimer’s disease (AD) perturbation (docking station PB cells). Our observation is consistent with emerging studies that exercise allows the trafficking of blood factors. Given the significance of FBXO32 and FOXO1 to ND and associated muscle integrity, our findings may explain, at least in part, the benefits of exercise on memory, associated gait, and balance perturbation acknowledged to herald the emergence of MCI.

scholarly journals Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation

2015 ◽  
Vol 35 (16) ◽  
pp. 2740-2751 ◽  
Author(s):  
Yi-Sheng Hou ◽  
Jun-Jie Guan ◽  
Hai-Dong Xu ◽  
Feng Wu ◽  
Rui Sheng ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Dependent Manner ◽  
Dopaminergic Cells ◽  
Protein Levels ◽  
Autophagy Induction ◽  
Ubiquitin Proteasome ◽  
Autophagic Degradation ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Autophagy Activity

Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson's disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells.

Chloroquine attenuates lipopolysaccharide-induced inflammatory responses through upregulation of USP25

2017 ◽  
Vol 95 (5) ◽  
pp. 481-491 ◽  
Author(s):  
Changyu Ding ◽  
Fangfang Li ◽  
Yupeng Long ◽  
Jiang Zheng
Keyword(s):  
Macrophage Activation ◽  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Ubiquitin Proteasome System ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Activation Of Transcription ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Ubiquitin Proteasome

Lipopolysaccharide (LPS) is a key pathogenic factor in sepsis, and its recognition by toll-like receptor 4 (TLR4) can activate two district signaling pathways, leading to activation of transcription factors including NF-κB and interferon regulatory factor 3 (IRF3). Chloroquine (CQ) has been shown to affect LPS–TLR4 colocalization and inhibit both MyD88-dependent and TRAM/TRIF-dependent pathways, though the mechanism involved is still poorly understood. Here, we found that the ubiquitin–proteasome system might be involved in this process. CQ increased USP25, a deubiquitinating enzyme, as well as mRNA and protein expression in a dose-dependent manner, which might to some degree be involved in CQ attenuation of LPS-induced macrophage activation. Overexpression of USP25 decreased LPS-induced inflammatory cytokines like TNF-α, IL-6, and IFN-β, while specific siRNA-mediated USP25 silencing increased TNF-α, IL-6, and IFN-β production and secretion. In addition, USP25 deletion strengthened mitogen-activated protein kinase (MAPKs) phosphorylation and IκB degradation. Moreover, USP25 interference increased NF-κB and IRF3 nuclear translocation. Taken together, our data demonstrated a new possible regulator of LPS-induced macrophage activation mediated by CQ, through upregulation of USP25.

scholarly journals β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Dongping Wang ◽  
Qi Zhang ◽  
Fenfen Li ◽  
Chan Wang ◽  
Changming Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Xenograft Model ◽  
Ubiquitin Proteasome System ◽  
Tumor Xenograft ◽  
Dependent Manner ◽  
Ubiquitin Proteasome ◽  
Novel Strategy ◽  
Hcc Cells

Abstract Background Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase (MAGUK) adaptor family of proteins and its deregulation has been implicated in the malignancy of several cancer types. Dlg5 was down-regulated in hepatocellular carcinoma (HCC) and lower Dlg5 expression was associated with poor survival of HCC patients. However, how to regulate Dlg5 remains largely unknown. Methods The co-immunoprecipitation assay was used to determine the interaction between Dlg5 and β-TrCP. The in vivo ubiquitination assay was performed to determine the regulation of Dlg5 by β-TrCP. CCK-8 and colony formation assay were implemented to detect the biological effect of Dlg5 on the growth of HCC cells in vitro. The effect of Dlg5 on HCC tumor growth in vivo was studied in a tumor xenograft model in mice. Results Here we report that Dlg5 is regulated by the ubiquitin proteasome system and depletion of either Cullin 1 or β-TrCP led to increased levels of Dlg5. β-TrCP regulated Dlg5 protein stability by targeting it for ubiquitination and subsequent destruction in a phosphorylation-dependent manner. We further demonstrated a crucial role of Ser730 in the non-canonical phosphodegron of Dlg5 in governing β-TrCP-mediated Dlg5 degradation. Importantly, failure to degrade Dlg5 significantly inhibited HCC cells proliferation both in vitro and in vivo. Conclusion Collectively, our finding provides a novel molecular mechanism for the negative regulation of Dlg5 by β-TRCP in HCC cells. It further suggests that preventing Dlg5 degradation could be a possible novel strategy for clinical treatment of HCC.

scholarly journals Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies

2020 ◽  
Vol 295 (30) ◽  
pp. 10138-10152 ◽  
Author(s):  
Janin Lautenschläger ◽  
Sara Wagner-Valladolid ◽  
Amberley D. Stephens ◽  
Ana Fernández-Villegas ◽  
Colin Hockings ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Protein Import ◽  
Protein Quality ◽  
Neurodegenerative Disorder ◽  
Mitochondrial Protein ◽  
Amyloid Β ◽  
Ubiquitin Proteasome System ◽  
Protein Homeostasis ◽  
Mitochondrial Inhibitors ◽  
Ubiquitin Proteasome

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin–proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

scholarly journals Clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins and preimplantation development

Development ◽  
2021 ◽  
Vol 148 (14) ◽  
Author(s):  
Akihito Morita ◽  
Yuhkoh Satouh ◽  
Hidetaka Kosako ◽  
Hisae Kobayashi ◽  
Akira Iwase ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Proteasome System ◽  
Maternal Plasma ◽  
Early Embryogenesis ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Selective Degradation ◽  
Kinase C ◽  
Ubiquitin Proteasome

ABSTRACT Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.

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