Evidence for the Direct Involvement of the Proteasome in the Proteolytic Processing of theAspergillus nidulansZinc Finger Transcription Factor PacC

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

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The p110 Isoform of the CDP/Cux Transcription Factor Accelerates Entry into S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2441-2455.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2441-2455 ◽

Cited By ~ 38

Author(s):

Laurent Sansregret ◽

Brigitte Goulet ◽

Ryoko Harada ◽

Brian Wilson ◽

Lam Leduy ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Cell Cycle Progression ◽

S Phase ◽

Proteolytic Processing ◽

Experimental Conditions ◽

Division Rate ◽

Cycle Progression ◽

Processing Event

ABSTRACT The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.

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Proteolytic processing of an Arabidopsis membrane-bound NAC transcription factor is triggered by cold-induced changes in membrane fluidity

Biochemical Journal ◽

10.1042/bj20091762 ◽

2010 ◽

Vol 427 (3) ◽

pp. 359-367 ◽

Cited By ~ 34

Author(s):

Pil Joon Seo ◽

Mi Jung Kim ◽

Jin-Su Song ◽

Youn-Sung Kim ◽

Hie-Joon Kim ◽

...

Keyword(s):

Transcription Factor ◽

Protein Stability ◽

Membrane Fluidity ◽

Calcium Influx ◽

Pathogen Resistance ◽

Membrane Lipid ◽

Proteolytic Processing ◽

Pharmacological Agents ◽

Proteolytic Activation ◽

Induced Changes

Changes in membrane fluidity are the earliest cellular events that occur in plant cells upon exposure to cold. This subsequently triggers physiological processes, such as calcium influx and reorganization of actin cytoskeletons, and induces expression of cold-responsive genes. The plasma-membrane-anchored NAC (NAM/ATAF/CUC) transcription factor NTL6 is of particular interest. Cold triggers proteolytic activation of the dormant NTL6 protein, which in turn elicits pathogen-resistance responses by inducing a small group of cold-inducible PR (pathogenesis-related) genes in Arabidopsis. In the present study, we show that proteolytic processing of NTL6 is regulated by cold-induced remodelling of membrane fluidity. NTL6 processing was stimulated rapidly by cold. The protein stability of NTL6 was also enhanced by cold. The effects of cold on NTL6 processing and protein stability were significantly reduced in cold-acclimatized plants, supporting the regulation of NTL6 processing by membrane fluidity. Consistent with this, although NTL6 processing was stimulated by pharmacological agents that reduce membrane fluidity and thus mimic cold, it was inhibited when plants were treated with a 18:3 unsaturated fatty acid, linolenic acid. In addition, the pattern of NTL6 processing was changed in Arabidopsis mutants with altered membrane lipid compositions. Assays employing chemicals that inhibit activities of the proteasome and proteases showed that NTL6 processing occurs via the regulated intramembrane proteolysis mechanism. Interestingly, a metalloprotease inhibitor blocked the NTL6 processing. These observations indicate that a metalloprotease activity is responsible for NTL6 processing in response to cold-induced changes in membrane fluidity.

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Transcriptional Regulation of BACE1, the β-Amyloid Precursor Protein β-Secretase, by Sp1

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.865-874.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 865-874 ◽

Cited By ~ 138

Author(s):

Michelle A. Christensen ◽

Weihui Zhou ◽

Hong Qing ◽

Anna Lehman ◽

Sjaak Philipsen ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Amyloid Precursor Protein ◽

Proteolytic Processing ◽

Precursor Protein ◽

Transcriptional Level ◽

Response Element ◽

App Processing ◽

Transcription Factor Sp1 ◽

Β Amyloid

ABSTRACT Proteolytic processing of the β-amyloid precursor protein (APP) at the β site is essential to generate Aβ. BACE1, the major β-secretase involved in cleaving APP, has been identified as a type 1 membrane-associated aspartyl protease. We have cloned a 2.1-kb fragment upstream of the human BACE1 gene and identified key regions necessary for promoter activity. BACE1 gene expression is controlled by a TATA-less promoter. The region of bp −619 to +46 is the minimal promoter to control the transcription of the BACE1 gene. Several putative cis-acting elements, such as a GC box, HSF-1, a PU box, AP1, AP2, and lymphokine response element, are found in the 5′ flanking region of the BACE1 gene. Transcriptional activation and gel shift assays demonstrated that the BACE1 promoter contains a functional Sp1 response element, and overexpression of the transcription factor Sp1 potentiates BACE gene expression and APP processing to generate Aβ. Furthermore, Sp1 knockout reduced BACE1 expression. These results suggest that BACE1 gene expression is tightly regulated at the transcriptional level and that the transcription factor Sp1 plays an important role in regulation of BACE1 to process APP generating Aβ in Alzheimer's disease.

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Controlled nuclear import of the transcription factor NTL6 reveals a cytoplasmic role of SnRK2.8 in the drought-stress response

Biochemical Journal ◽

10.1042/bj20120244 ◽

2012 ◽

Vol 448 (3) ◽

pp. 353-363 ◽

Cited By ~ 61

Author(s):

Mi Jung Kim ◽

Mi-Jeong Park ◽

Pil Joon Seo ◽

Jin-Su Song ◽

Hie-Joon Kim ◽

...

Keyword(s):

Transcription Factor ◽

Drought Stress ◽

Transgenic Plants ◽

Drought Resistance ◽

Nuclear Import ◽

Proteolytic Processing ◽

Transcriptional Responses ◽

Resistance Response ◽

Proteolytic Activation

Controlled proteolytic activation of membrane-anchored transcription factors provides an adaptation strategy that guarantees rapid transcriptional responses to abrupt environmental stresses in both animals and plants. NTL6 is a plant-specific NAC [NAM/ATAF1/2/CUC2] transcription factor that is expressed as a dormant plasma membrane-associated form in Arabidopsis. Proteolytic processing of NTL6 is triggered by abiotic stresses and ABA (abscisic acid). In the present study, we show that NTL6 is linked directly with SnRK (Snf1-related protein kinase) 2.8-mediated signalling in inducing a drought-resistance response. SnRK2.8 phosphorylates NTL6 primarily at Thr142. NTL6 phosphorylation by SnRK2.8 is required for its nuclear import. Accordingly, a mutant NTL6 protein, in which Thr142 was mutated to an alanine, was poorly phosphorylated and failed to enter the nucleus. In accordance with the role of SnRK2.8 in drought-stress signalling, transgenic plants overproducing either NTL6 or its active form 6ΔC (35S:NTL6 and 35S:6ΔC) exhibited enhanced resistance to water-deficit conditions such as those overproducing SnRK2.8 (35S:SnRK2.8). In contrast, NTL6 RNAi (RNA interference) plants were susceptible to dehydration as observed in the SnRK2.8-deficient snrk2.8-1 mutant. Furthermore, the dehydration-resistant phenotype of 35S:NTL6 transgenic plants was compromised in 35S:NTL6 X snrk2.8-1 plants. These observations indicate that SnRK2.8-mediated protein phosphorylation, in addition to a proteolytic processing event, is important for NTL6 function in inducing a drought-resistance response.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21010327 ◽

2020 ◽

Vol 21 (1) ◽

pp. 327 ◽

Cited By ~ 8

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.

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Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00247-09 ◽

2010 ◽

Vol 9 (6) ◽

pp. 943-951 ◽

Cited By ~ 13

Author(s):

Masafumi Nishizawa ◽

Mirai Tanigawa ◽

Michio Hayashi ◽

Tatsuya Maeda ◽

Yoshiaki Yazaki ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Cellular Response ◽

Proteolytic Processing ◽

Yeast Cells ◽

Nuclear Accumulation ◽

Alkaline Stress ◽

Content Type ◽

Its Gene ◽

Alkaline Conditions

ABSTRACT The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.

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A new kind of membrane-tethered eukaryotic transcription factor that shares an auto-proteolytic processing mechanism with bacteriophage tail-spike proteins

Journal of Cell Science ◽

10.1242/jcs.133231 ◽

2013 ◽

Vol 126 (22) ◽

pp. 5247-5258 ◽

Cited By ~ 6

Author(s):

H. Senoo ◽

T. Araki ◽

M. Fukuzawa ◽

J. G. Williams

Keyword(s):

Transcription Factor ◽

Proteolytic Processing ◽

Eukaryotic Transcription ◽

Tail Spike ◽

Processing Mechanism ◽

Spike Proteins

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NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6089 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6089-6101 ◽

Cited By ~ 64

Author(s):

R I Scheinman ◽

A A Beg ◽

A S Baldwin

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Phorbol Esters ◽

Interleukin 1 ◽

Binding Activity ◽

Proteolytic Processing ◽

Cellular Growth ◽

Nf Kappa B ◽

Dna Binding Activity ◽

Multiple Copies

NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappa B p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an I kappa B-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappa B activity distinct from the classic I kappa B pathway.

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Specificity Determinants of Proteolytic Processing of Aspergillus PacC Transcription Factor Are Remote from the Processing Site, and Processing Occurs in Yeast If pH Signalling Is Bypassed

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1390 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1390-1400 ◽

Cited By ~ 55

Author(s):

José-Manuel Mingot ◽

Joan Tilburn ◽

Eliecer Diez ◽

Elaine Bignell ◽

Margarita Orejas ◽

...

Keyword(s):

Transcription Factor ◽

Functional Form ◽

Ph Regulation ◽

Structural Features ◽

Proteolytic Processing ◽

Type I ◽

Independent Manner ◽

Mutant Proteins ◽

Processing Site ◽

Specificity Determinants

ABSTRACT The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of “neutrality-mimicking” pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues ∼252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur inSaccharomyces cerevisiae.

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