scholarly journals Degradation of the Tumor Suppressor PDCD4 Is Impaired by the Suppression of p62/SQSTM1 and Autophagy

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 218 ◽  
Author(s):  
M. Manirujjaman ◽  
Iwata Ozaki ◽  
Yuzo Murata ◽  
Jing Guo ◽  
Jinghe Xia ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Proteasome Inhibitor ◽  
Transcriptional Control ◽  
Ubiquitin Proteasome System ◽  
Cellular Functions ◽  
Protein Levels ◽  
Sequestosome 1 ◽  
Huh7 Cells ◽  
Programmed Cell Death 4 ◽  
Pdcd4 Protein

PDCD4 (programmed cell death 4) is a tumor suppressor that plays a crucial role in multiple cellular functions, such as the control of protein synthesis and transcriptional control of some genes, the inhibition of cancer invasion and metastasis. The expression of this protein is controlled by synthesis, such as via transcription and translation, and degradation by the ubiquitin-proteasome system. The mitogens, known as tumor promotors, EGF (epidermal growth factor) and TPA (12-O-tetradecanoylphorbol-13-acetate) stimulate the degradation of PDCD4 protein. However, the whole picture of PDCD4 degradation mechanisms is still unclear, we therefore investigated the relationship between PDCD4 and autophagy. The proteasome inhibitor MG132 and the autophagy inhibitor bafilomycin A1 were found to upregulate the PDCD4 levels. PDCD4 protein levels increased synergistically in the presence of both inhibitors. Knockdown of p62/SQSTM1 (sequestosome-1), a polyubiquitin binding partner, also upregulated the PDCD4 levels. P62 and LC3 (microtubule-associated protein 1A/1B-light chain 3)-II were co-immunoprecipitated by an anti-PDCD4 antibody. Colocalization particles of PDCD4, p62 and the autophagosome marker LC3 were observed and the colocalization areas increased in the presence of autophagy and/or proteasome inhibitor(s) in Huh7 cells. In ATG (autophagy related) 5-deficient Huh7 cells in which autophagy was impaired, the PDCD4 levels were increased at the basal levels and upregulated in the presence of autophagy inhibitors. Based on the above findings, we concluded that after phosphorylation in the degron and ubiquitination, PDCD4 is degraded by both the proteasome and autophagy systems.

scholarly journals NRF3-POMP-20S Proteasome Assembly Axis Promotes Cancer Development via Ubiquitin-Independent Proteolysis of p53 and Retinoblastoma Protein

2020 ◽  
Vol 40 (10) ◽  
Author(s):  
Tsuyoshi Waku ◽  
Nanami Nakamura ◽  
Misaki Koji ◽  
Hidenori Watanabe ◽  
Hiroki Katoh ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Cells ◽  
Proteasome Inhibitor ◽  
Rectal Adenocarcinoma ◽  
26S Proteasome ◽  
Cancer Development ◽  
20S Proteasome ◽  
Cancer Cell Growth ◽  
Protein Levels ◽  
Cancer Tissues

ABSTRACT Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome have been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Here, we show that the cap ’n’ collar (CNC) family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and that 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and retinoblastoma (Rb) proteins. The NRF3 gene is highly expressed in many cancer tissues and cell lines and is important for cancer cell growth. In cancer cells, NRF3 upregulates the assembly of the 20S proteasome by directly inducing the gene expression of the 20S proteasome maturation protein POMP. Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increases p53 activities for tumor suppression, including cell cycle arrest and induction of apoptosis. Furthermore, protein stability and cell viability assays using two distinct proteasome inhibitor anticancer drugs, the 20S proteasome inhibitor bortezomib and the ubiquitin-activating enzyme E1 inhibitor TAK-243, show that the upregulation of the NRF3-POMP axis leads to ubiquitin-independent proteolysis of p53 and Rb and to impaired sensitivity to bortezomib but not TAK-243. More importantly, the NRF3-POMP axis supports tumorigenesis and metastasis, with higher NRF3/POMP expression levels correlating with poor prognoses in patients with colorectal or rectal adenocarcinoma. These results suggest that the NRF3-POMP-20S proteasome assembly axis is significant for cancer development via ubiquitin-independent proteolysis of tumor suppressor proteins.

Targeting the Ubiquitin-proteasome System for the Treatment of Multiple Myeloma and Other Human Diseases

2010 ◽  
Vol 2 ◽  
pp. CMT.S2889
Author(s):  
Klaus Podar ◽  
Kenneth C. Anderson
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Ubiquitin Proteasome System ◽  
Hematologic Malignancies ◽  
Mechanisms Of Action ◽  
Human Diseases ◽  
Cellular Functions ◽  
Adverse Side Effects ◽  
Ubiquitin Proteasome ◽  
Novel Approaches

The ubiquitin-proteasome-degradation system plays a key role in multiple cellular functions. Its deregulation is associated with the initiation and progression of human diseases including not only solid and hematologic malignancies but also neurologic and autoimmune disorders. This article discusses several novel mechanistic aspects of the ubiquitin-proteasome system. Moreover, it focuses on the development, mechanisms of action, and clinical experience with Bortezomib, the first in-class-proteasome inhibitor to enter the clinics. Finally, it summarizes novel approaches to specifically target distinct components within the highly complex and dynamic ubiquitin-proteasome machinery to ultimately further increase drug activity, as well as reduce drug resistance and adverse side effects.

scholarly journals Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits

2011 ◽  
Vol 286 (22) ◽  
pp. 19191-19203 ◽  
Author(s):  
Makoto Miyazawa ◽  
Erika Tashiro ◽  
Hirotake Kitaura ◽  
Hiroshi Maita ◽  
Hiroo Suto ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Proteasome Inhibitor ◽  
Binding Protein ◽  
Ubiquitin Proteasome System ◽  
Unfolded Proteins ◽  
Protein Levels ◽  
Mutual Regulation ◽  
Ubiquitin Proteasome ◽  
Transformation Activity ◽  
Regulate Protein

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

scholarly journals The Autophagy Pathway: A Critical Route in the Disposal of Alpha 1-Antitrypsin Aggregates That Holds Many Mysteries

2021 ◽  
Vol 22 (4) ◽  
pp. 1875
Author(s):  
Celine Leon ◽  
Marion Bouchecareilh
Keyword(s):  
Liver Transplantation ◽  
Liver Disease ◽  
Therapeutic Option ◽  
Ubiquitin Proteasome System ◽  
Therapeutic Benefit ◽  
Cellular Functions ◽  
Precise Control ◽  
New Therapeutic Approaches ◽  
Alpha 1 Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin

The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin–proteasome system (UPS) or the autophagy–lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.

Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2797-2805 ◽  
Author(s):  
Feng-Ting Liu ◽  
Samir G. Agrawal ◽  
John G. Gribben ◽  
Hongtao Ye ◽  
Ming-Qing Du ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Resistant Cell ◽  
Protein Levels ◽  
Bax Protein ◽  
Degradation Activity ◽  
Potent Drug ◽  
Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

scholarly journals Reprimo (RPRM) Is a Novel Tumor Suppressor in Pituitary Tumors and Regulates Survival, Proliferation, and Tumorigenicity

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 2963-2973 ◽  
Author(s):  
Mei Xu ◽  
Aaron J. Knox ◽  
Katherine A. Michaelis ◽  
Katja Kiseljak-Vassiliades ◽  
Bette K. Kleinschmidt-DeMasters ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tumor Suppressor ◽  
Cellular Stress ◽  
Pituitary Tumors ◽  
Tumor Formation ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Null Cell ◽  
Human Pituitary ◽  
Protein Levels

Reprimo (RPRM), initially identified as a downstream effector of p53-induced cell cycle arrest at G2/M, is a putative tumor suppressor silenced in some types of cancer. In microarrays, the RPRM transcript was repressed 26-fold in gonadotrope (null cell) human pituitary tumors compared with normal pituitary but in the absence of changes in p53. Inhibition of RPRM mRNA was confirmed by RT-PCR in all gonadotrope tumors, most GH samples, and variably in other tumor types. Human pituitary tumors showed no evidence of abnormal promoter hypermethylation as a mechanism of RPRM repression. RPRM stable expression in gonadotrope (LβT2) and GH (GH3) pituitary cells resulted in decreased rates of cell proliferation by 55 and 30%, respectively; however, RPRM reexpression did not alter G2/M transition. In addition, RPRM increased rates of apoptosis in response to growth factor deprivation as assessed by caspase-3 cleavage and nuclear condensation. Clonagenic assays showed a 5.3- and 3.7-fold suppression of colony growth in RPRM-overexpressing LβT2 and GH3 cells, respectively, supporting its role as a tumor suppressor. In cells stably expressing RPRM mRNA, protein levels were actively suppressed due to rapid degradation through ubiquitination and proteasomal targeting. Growth factor withdrawal, as a model of cellular stress, stabilized RPRM protein levels. Together these data suggest that RPRM is transiently up-regulated at a posttranscriptional level in times of cellular stress to restrict cell survival, proliferation, and tumor formation. When RPRM is silenced as in human pituitary tumors, unrestrained growth and tumor progression may occur.

scholarly journals Alteration of transporter activities in the epididymides of infertile initial segment-specific Pten knockout mice†

2018 ◽  
Vol 99 (3) ◽  
pp. 536-545 ◽  
Author(s):  
Bingfang Xu ◽  
Stephen D Turner ◽  
Barry T Hinton
Keyword(s):  
Male Fertility ◽  
Initial Segment ◽  
Molecular Mechanisms ◽  
Fluorescein Isothiocyanate ◽  
Proximal Region ◽  
Sperm Maturation ◽  
Cellular Functions ◽  
Permeability Characteristics ◽  
Protein Levels ◽  
Tensin Homolog

Abstract A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate–dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.

scholarly journals Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response

2013 ◽  
Vol 45 (4) ◽  
pp. 127-137 ◽  
Author(s):  
Lingchen Fu ◽  
Michael S. Kilberg
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Translational Control ◽  
Mammalian Cells ◽  
Transcriptional Control ◽  
Present Report ◽  
Binding Activity ◽  
Maximal Response ◽  
Protein Levels ◽  
Amino Acid Response

Mammalian cells respond to amino acid deprivation through multiple signaling pathways referred to as the amino acid response (AAR). Transcription factors mediate the AAR after their activation by several mechanisms; examples include translational control (activating transcription factor 4, ATF4), phosphorylation (p-cJUN), and transcriptional control (ATF3). ATF4 induces ATF3 transcription through a promoter-localized C/EBP-ATF response element (CARE). The present report characterizes an ATF/CRE site upstream of the CARE that also contributes to AAR-induced ATF3 transcription. ATF4 binds to the ATF/CRE and CARE sequences and both are required for a maximal response to ATF4 induction. ATF3, which antagonizes ATF4 and represses its own gene, also exhibited binding activity to the ATF/CRE and CARE sequences. The AAR resulted in elevated total cJUN and p-cJUN protein levels and both forms exhibited binding activity to the ATF/CRE and CARE ATF3 sequences. Knockdown of AAR-enhanced cJUN expression blocked induction of the ATF3 gene and mutation of either the ATF/CRE or the CARE site prevented the cJUN-dependent increase in ATF3-driven luciferase activity. The results indicate that both increased cJUN and the cis-acting ATF/CRE sequence within the ATF3 promoter contribute to the transcriptional activation of the gene during the AAR.

scholarly journals SRSF3 promotes pluripotency through Nanog mRNA export and coordination of the pluripotency gene expression program

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Madara Ratnadiwakara ◽  
Stuart K Archer ◽  
Craig I Dent ◽  
Igor Ruiz De Los Mozos ◽  
Traude H Beilharz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cellular Functions ◽  
Protein Levels ◽  
The Core ◽  
Molecular Events ◽  
Coordinate Gene Expression ◽  
Pluripotency Gene ◽  
Steady State Levels ◽  
Expression Program ◽  
Pluripotency Genes

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

scholarly journals Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription Factors

2006 ◽  
Vol 17 (2) ◽  
pp. 585-597 ◽  
Author(s):  
Fang Liu ◽  
Nabendu Pore ◽  
Mijin Kim ◽  
K. Ranh Voong ◽  
Melissa Dowling ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
Histone Deacetylases ◽  
Targeted Mutagenesis ◽  
Cellular Functions ◽  
Specific Expression ◽  
Protein Levels ◽  
Histone Deacetylase 4 ◽  
First Time

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.

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