scholarly journals p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Senthil K Radhakrishnan ◽  
Willem den Besten ◽  
Raymond J Deshaies
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Proteasome Inhibition ◽  
Active Species ◽  
Proteolytic Processing ◽  
Evolutionarily Conserved ◽  
Cytosolic Side ◽  
Homeostatic Response ◽  
Bounce Back ◽  
Proteasome Gene

Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., ‘bounce-back’) of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol.

scholarly journals p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

2013 ◽  
Author(s):  
Senthil K Radhakrishnan ◽  
Willem den Besten ◽  
Raymond J Deshaies
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Proteasome Inhibition ◽  
Active Species ◽  
Proteolytic Processing ◽  
Evolutionarily Conserved ◽  
Cytosolic Side ◽  
Homeostatic Response ◽  
Bounce Back ◽  
Proteasome Gene

Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e. 'bounce-back') of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. Article now published as eLife 2014;3:e01856 http://dx.doi.org/10.7554/eLife.01856

scholarly journals Disabling the Protease DDI2 Attenuates the Transcriptional Activity of NRF1 and Potentiates Proteasome Inhibitor Cytotoxicity

2020 ◽  
Vol 21 (1) ◽  
pp. 327 ◽  
Author(s):  
Amy Northrop ◽  
Janakiram R. Vangala ◽  
Alex Feygin ◽  
Senthil K. Radhakrishnan
Keyword(s):  
Transcription Factor ◽  
Proteasome Inhibitor ◽  
Mammalian Cells ◽  
Active Form ◽  
Cancer Cell Line ◽  
Proteasome Inhibition ◽  
Proteolytic Processing ◽  
Related Factor ◽  
Compensatory Mechanism ◽  
Proteotoxic Stress

Proteasome inhibition is used therapeutically to induce proteotoxic stress and trigger apoptosis in cancer cells that are highly dependent on the proteasome. As a mechanism of resistance, inhibition of the cellular proteasome induces the synthesis of new, uninhibited proteasomes to restore proteasome activity and relieve proteotoxic stress in the cell, thus evading apoptosis. This evolutionarily conserved compensatory mechanism is referred to as the proteasome-bounce back response and is orchestrated in mammalian cells by nuclear factor erythroid derived 2-related factor 1 (NRF1), a transcription factor and master regulator of proteasome subunit genes. Upon synthesis, NRF1 is cotranslationally inserted into the endoplasmic reticulum (ER), then is rapidly retrotranslocated into the cytosol and degraded by the proteasome. In contrast, during conditions of proteasome inhibition or insufficiency, NRF1 escapes degradation, is proteolytically cleaved by the aspartyl protease DNA damage inducible 1 homolog 2 (DDI2) to its active form, and enters the nucleus as an active transcription factor. Despite these insights, the cellular compartment where the proteolytic processing step occurs remains unclear. Here we further probed this pathway and found that NRF1 can be completely retrotranslocated into the cytosol where it is then cleaved and activated by DDI2. Furthermore, using a triple-negative breast cancer cell line MDA-MB-231, we investigated the therapeutic utility of attenuating DDI2 function. We found that DDI2 depletion attenuated NRF1 activation and potentiated the cytotoxic effects of the proteasome inhibitor carfilzomib. More importantly, expression of a point-mutant of DDI2 that is protease-dead recapitulated these effects. Taken together, our results provide a strong rationale for a combinational therapy that utilizes inhibition of the proteasome and the protease function of DDI2. This approach could expand the repertoire of cancer types that can be successfully treated with proteasome inhibitors in the clinic.

scholarly journals The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Shun Koizumi ◽  
Taro Irie ◽  
Shoshiro Hirayama ◽  
Yasuyuki Sakurai ◽  
Hideki Yashiroda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Mammalian Cells ◽  
Proteasome Inhibition ◽  
Cancer Therapies ◽  
Aspartyl Protease ◽  
Wild Type ◽  
Proteasome Gene

In response to proteasome dysfunction, mammalian cells upregulate proteasome gene expression by activating Nrf1. Nrf1 is an endoplasmic reticulum-resident transcription factor that is continually retrotranslocated and degraded by the proteasome. Upon proteasome inhibition, Nrf1 escapes degradation and is cleaved to become active. However, the processing enzyme for Nrf1 remains obscure. Here we show that the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is required to cleave and activate Nrf1. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form of Nrf1, resulting in poor upregulation of proteasomes in response to proteasome inhibition. These defects were restored by adding back wild-type DDI2 but not protease-defective DDI2. Our results provide a clue for blocking compensatory proteasome synthesis to improve cancer therapies targeting proteasomes.

scholarly journals Transcription Factor Nrf1 Mediates the Proteasome Recovery Pathway after Proteasome Inhibition in Mammalian Cells

2010 ◽  
Vol 38 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Senthil K. Radhakrishnan ◽  
Candy S. Lee ◽  
Patrick Young ◽  
Anne Beskow ◽  
Jefferson Y. Chan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Proteasome Inhibition

scholarly journals The p97-UBXN1 complex regulates aggresome formation

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.

scholarly journals The p97-UBXN1 complex regulates aggresome formation

2021 ◽  
pp. jcs.254201
Author(s):  
Sirisha Mukkavalli ◽  
Jacob Aaron Klickstein ◽  
Betty Ortiz ◽  
Peter Juo ◽  
Malavika Raman
Keyword(s):  
Protein Aggregation ◽  
Mammalian Cells ◽  
Inclusion Bodies ◽  
Proteasome Inhibition ◽  
Adaptor Proteins ◽  
Cellular Homeostasis ◽  
Aaa Atpase ◽  
Evolutionarily Conserved ◽  
Aggresome Formation

The recognition and disposal of misfolded proteins is 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. The p97 AAA-ATPase in combination with adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the proteasome or autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes, membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates aggresome formation and clearance and identify a novel role for the adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to aggresomes and UBXN1 knockout cells are unable to form aggresomes. Loss of p97-UBXN1 results in increased Huntingtin polyQ inclusion bodies 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-UBXN1 in the disposal of protein aggregates.

scholarly journals Nelfinavir Inhibits the TCF11/Nrf1-Mediated Proteasome Recovery Pathway in Multiple Myeloma

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1065 ◽  
Author(s):  
Dominika Fassmannová ◽  
František Sedlák ◽  
Jindřich Sedláček ◽  
Ivan Špička ◽  
Klára Grantz Šašková
Keyword(s):  
Multiple Myeloma ◽  
Protein Level ◽  
3D Structure ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteolytic Processing ◽  
Hiv Protease ◽  
Hiv Protease Inhibitors ◽  
Development Of Resistance ◽  
Proteasome Gene

Proteasome inhibitors are the backbone of multiple myeloma therapy. However, disease progression or early relapse occur due to development of resistance to the therapy. One important cause of resistance to proteasome inhibition is the so-called bounce-back response, a recovery pathway driven by the TCF11/Nrf1 transcription factor, which activates proteasome gene re-synthesis upon impairment of the proteasome function. Thus, inhibiting this recovery pathway potentiates the cytotoxic effect of proteasome inhibitors and could benefit treatment outcomes. DDI2 protease, the 3D structure of which resembles the HIV protease, serves as the key player in TCF11/Nrf1 activation. Previous work found that some HIV protease inhibitors block DDI2 in cell-based experiments. Nelfinavir, an oral anti-HIV drug, inhibits the proteasome and/or pAKT pathway and has shown promise for treatment of relapsed/refractory multiple myeloma. Here, we describe how nelfinavir inhibits the TCF11/Nrf1-driven recovery pathway by a dual mode of action. Nelfinavir decreases the total protein level of TCF11/Nrf1 and inhibits TCF11/Nrf1 proteolytic processing, likely by interfering with the DDI2 protease, and therefore reduces the TCF11/Nrf1 protein level in the nucleus. We propose an overall mechanism that explains nelfinavir’s effectiveness in the treatment of multiple myeloma.

scholarly journals Switching on pluripotency: a perspective on the biological requirement of Nanog

2011 ◽  
Vol 366 (1575) ◽  
pp. 2222-2229 ◽  
Author(s):  
Thorold W. Theunissen ◽  
José C. R. Silva
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Induced Pluripotency ◽  
Somatic Cell Reprogramming ◽  
Evolutionarily Conserved

Pluripotency is a transient cellular state during early development which can be recreated in vitro by direct reprogramming. The molecular mechanisms driving entry into and exit from the pluripotent state are the subject of intense research interest. Here, we review the role of the homeodomain-containing transcription factor Nanog in mammalian embryology and induced pluripotency. Nanog was originally thought to be confined to the maintenance of pluripotency, but recent insights from genetic studies uncovered a new biological function. Embryonic stem cells deficient in Nanog alleles are more prone to differentiate but do not lose pluripotency per se . Instead, Nanog is transiently required for the specification of the naive pluripotent epiblast and development of primordial germ cells. Nanog is also essential to finalize somatic cell reprogramming during induction of pluripotency. We propose that this unique transcription factor acts as a molecular switch to turn on the naive pluripotent programme in mammalian cells. In this context, the capacity of Nanog to resist differentiation can be regarded as recapitulation of effects normally associated with the specification of pluripotency. Pertinent questions are how Nanog specifies naive pluripotency and whether this mechanism is evolutionarily conserved.

Apoptosis and development

2003 ◽  
Vol 39 ◽  
pp. 11-24 ◽  
Author(s):  
Justin V McCarthy
Keyword(s):  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Cell Number ◽  
Model Organisms ◽  
Biological Processes ◽  
Nematode Caenorhabditis Elegans ◽  
Apoptotic Pathway ◽  
Genetic Pathways ◽  
Evolutionarily Conserved ◽  
Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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