Inhibition of mitochondrial translation overcomes venetoclax resistance in AML through activation of the integrated stress response

Venetoclax is a specific B cell lymphoma 2 (BCL-2) inhibitor with promising activity against acute myeloid leukemia (AML), but its clinical efficacy as a single agent or in combination with hypomethylating agents (HMAs), such as azacitidine, is hampered by intrinsic and acquired resistance. Here, we performed a genome-wide CRISPR knockout screen and found that inactivation of genes involved in mitochondrial translation restored sensitivity to venetoclax in resistant AML cells. Pharmacologic inhibition of mitochondrial protein synthesis with antibiotics that target the ribosome, including tedizolid and doxycycline, effectively overcame venetoclax resistance. Mechanistic studies showed that both tedizolid and venetoclax suppressed mitochondrial respiration, with the latter demonstrating inhibitory activity against complex I [nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase] of the electron transport chain (ETC). The drugs cooperated to activate a heightened integrated stress response (ISR), which, in turn, suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. Combination treatment with tedizolid and venetoclax was superior to either agent alone in reducing leukemic burden in mice engrafted with treatment-resistant human AML. The addition of tedizolid to azacitidine and venetoclax further enhanced the killing of resistant AML cells in vitro and in vivo. Our findings demonstrate that inhibition of mitochondrial translation is an effective approach to overcoming venetoclax resistance and provide a rationale for combining tedizolid, azacitidine, and venetoclax as a triplet therapy for AML.

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Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

Cell Death and Disease ◽

10.1038/s41419-020-03324-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ai-Ling Tian ◽

Qi Wu ◽

Peng Liu ◽

Liwei Zhao ◽

Isabelle Martins ◽

...

Keyword(s):

Stress Response ◽

Immune Responses ◽

Initiation Factor ◽

Integrated Stress Response ◽

Serine Residue ◽

Eif2α Phosphorylation ◽

Lysosomotropic Agents ◽

Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

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The BH3 Mimetic, ABT-737, Is Effective Against Bcl-2 Overexpressing Lymphoid Tumors.

Blood ◽

10.1182/blood.v108.11.826.826 ◽

2006 ◽

Vol 108 (11) ◽

pp. 826-826 ◽

Cited By ~ 1

Author(s):

Kylie D. Mason ◽

Cassandra J. Vandenberg ◽

Mark F. van Delft ◽

Andrew H. Wei ◽

Suzanne Cory ◽

...

Keyword(s):

Cell Lines ◽

Genetic Manipulation ◽

Single Agent ◽

B Cell Lymphoma ◽

Cell Types ◽

Clinical Samples ◽

Bh3 Mimetic ◽

Mouse Lymphoma

Abstract Lymphoid tumors often respond poorly to conventional cytotoxics, a common cause being their impaired sensitivity to apoptosis, such as that caused by Bcl-2 overexpression. A strategy to overcoming this is to use mimics of the natural antagonists of pro-survival Bcl-2, the BH3 only proteins. A promising BH3 mimetic is ABT-737, which targets Bcl-2 and closely related pro-survival proteins. We evaluated its potential utility by testing it on cell lines, clinical samples and on a relevant mouse lymphoma model. We assessed the sensitivity of B cell lymphoma cell lines and primary CLL samples to ABT-737, either alone or in combination. To ascertain its efficacy in vivo, we utilized a mouse model based on the Eμ-myc tumor that is readily transplantable and amenable to genetic manipulation. When syngeneic recipient mice were inoculated with tumors, they develop widespread lymphoma, fatal unless treated by agents such as cyclophosphamide. We found that ABT-737, on its own, was cytotoxic only to a subset of cell lines and primary CLL samples. However, it can synergize potently with agents such as dexamethasone, suggesting that this agent might be useful in combination with currently used chemotherapeutics. In the Eμ myc mouse lymphoma model, treatment with ABT-737 alone did not control the disease as multiple independently derived tumors proved refractory to treatment with this agent. However, ABT-737 was partially effective as a single agent for treating bitransgenic tumors derived from crosses of the Eμmyc and Eμ-bcl-2 transgenic mice. ABT-737 therapy prolonged the survival of recipient mice transplanted with tumors from 30 to 60 days. When combined with a low dose of cyclophosphamide (50mg/kg), long term stable remissions were achieved, which were sustained even longer than control mice treated with much higher doses of cyclophosphamide (300mg/kg). We found that ABT-737 was well tolerated as a single agent and when combined with low doses of cytotoxics such as cyclophosphamide. Thus, ABT-737 may prove to be efficacious for those tumors highly dependent on Bcl-2 for their survival. We found that despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737 as a single agent. We show that this resistance reflects its inability to target another pro-survival relative Mcl-1. Down-regulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in the Eμmyc/bcl-2 bitransgenic mouse lymphoma model conferred marked resistance as mice transplanted with such tumors died as rapidly as the untreated counterparts. However, enhanced Bcl-2 overexpression on these tumors had little impact on the in vivo response, suggesting that ABT-737 can be utilized even when Bcl-2 is markedly overexpressed. ABT-737 appears to be a promising agent for the clinic. It potently sensitizes certain lymphoid tumors to conventional cytotxics in vitro. The synergy observed between dexamethasone and ABT-737 on some lymphoid lines in culture suggests that it is attractive for clinical testing. Encouragingly, ABT-737 appeared efficacious in vivo against Bcl-2 overexpressing tumors when combined with a reduced dose of cyclophosphamide, suggesting that it will be useful for treating even those Bcl-2-overexpressing tumors that are normally highly chemoresistant.

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HCD122, an Antagonist Human Anti-CD40 Monoclonal Antibody, Enhances Efficacy of CHOP in Tumor Xenograft Model of Human Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v110.11.528.528 ◽

2007 ◽

Vol 110 (11) ◽

pp. 528-528 ◽

Cited By ~ 1

Author(s):

Mohammad Luqman ◽

Ssucheng J. Hsu ◽

Matthew Ericson ◽

Sha Klabunde ◽

Seema Kantak

Keyword(s):

Monoclonal Antibody ◽

Clinical Trials ◽

Tumor Growth ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract HCD122 (formerly known as CHIR-12.12), is a fully human anti-CD40 monoclonal antibody (mAb) currently in Phase I clinical trials for treatment of chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). An IgG1 antibody selected for its potency as an antagonist of the CD40 signaling pathway, HCD122 both inhibits CD40/CD40L-stimulated growth of lymphoma cells ex vivo, and mediates highly effective Antibody Dependent Cell-mediated Cytotoxicity (ADCC) in vitro. As a single agent, HCD122 exhibits potent anti-tumor activity in vivo, in preclinical models of MM, Hodgkin’s lymphoma, Burkitt’s lymphoma, mantle cell lymphoma and diffused large B-cell lymphoma (DLBCL). Although several therapeutic antibodies approved for treatment of Non-Hodgkin’s Lymphoma have clinical activity as single agents, combining these antibodies with standard-of-care chemotherapeutic regimens such as CHOP (cytoxan, vincristine, doxorubicin and prednisone) is proving optimal for both increasing response rates and extending survival, and antibodies currently in clinical development are likely to be used in combination therapies in the future. Therefore the studies reported here examine the effects of combining HCD122 with CHOP, the standard for treatment of high grade NHL, in in vitro and in vivo models of DLBCL. In the xenograft RL model of DLBCL, HCD122 administered intraperitoneally weekly at 1 mg/kg as a single agent, or in combination with CHOP (H-CHOP), and CHOP alone all significantly reduced tumor growth at day 25 when compared to treatment with huIgG1 control antibody (P<0.001). However, tumor growth delay (time to reach tumor size of 500 mm3) was significantly longer for H-CHOP (17.5 days), than for CHOP (8 days) or HCD122 (6 days) (p < 0.001). No toxicity was observed with the H-CHOP combination. Interestingly, at the end of the study (day 35), reduction in tumor growth was significantly greater in the treatment group that received H-CHOP than the groups that received either 10 mg/kg Rituxan plus CHOP (R-CHOP) (p < 0.05) or CHOP alone (p < 0.001). These data show that in this model, treatment with the combination H-CHOP results in greater anti-tumor efficacy than with either modality alone or R-CHOP. We have observed that in vitro, exposure to CD40 Ligand (CD40L) results in aggregation of DLBCL cells, and postulate that interfering with the ability of cancer cells to adhere and interact with each other and their microenvironment may potentiate the effect of chemotherapeutics. To elucidate the mechanism by which the combination of HCD122 and CHOP enhanced efficacy in vivo, we developed an in vitro system to examine the effects of HCD122 on the expression of adhesion molecules in the RL and SU-DHL-4 cell lines. In these studies, HCD122 inhibited CD40L-induced expression of CD54, CD86 and CD95 in both cell lines, as well as aggregation of SU-DHL-4 cells. The combined effect of each of the components of CHOP with HCD122 in three-dimensional spheroid cultures is currently under investigation. These data provide a therapeutic rationale for combination of HCD122 with CHOP in DLBCL clinical trials.

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eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

10.1101/2020.12.28.424567 ◽

2020 ◽

Author(s):

Michael Schoof ◽

Morgane Boone ◽

Lan Wang ◽

Rosalie Lawrence ◽

Adam Frost ◽

...

Keyword(s):

Stress Response ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Α Subunit ◽

Rocking Motion

AbstractThe integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

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eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽

10.7554/elife.65703 ◽

2021 ◽

Vol 10 ◽

Author(s):

Michael Schoof ◽

Morgane Boone ◽

Lan Wang ◽

Rosalie Lawrence ◽

Adam Frost ◽

...

Keyword(s):

Stress Response ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Α Subunit ◽

Rocking Motion

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

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Active Combination Therapy of Bortezomib (Velcade) and Ibritumomab Tiuxetan (Zevalin) in an In Vivo Diffuse Large B-Cell Lymphoma Model.

Blood ◽

10.1182/blood.v106.11.2406.2406 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2406-2406 ◽

Cited By ~ 3

Author(s):

Milena Pervan ◽

Jeffrey Calimlim ◽

Dan Matso ◽

Jonathan W. Said ◽

William H. McBride ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Ibritumomab Tiuxetan ◽

Treatment Groups ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Chemotherapy-resistant diffuse large B-cell lymphomas (DLBCL) pose an unresolved clinical problem and most patients die of their disease within months. The goal of this study was to test a novel, non-chemotherapy containing regimen for Non Hodgkin’s lymphomas, including DLBCL, by combining the proteasome inhibitor bortezomib (PS-341, VELCADE®) with radio-immunotherapy using ibritumomab tiuxetan (Zevalin™). Previously, we have shown that the combination of bortezomib and rituximab is an active and at least additive regimen in an in vitro and in vivo DLBCL model. These data supported an ongoing clinical trial using this combination in patients with low grade NHL. In addition, Pervan et al. identified the proteasome as a novel, sensitive target for ionizing radiation (Mol Cancer Res. 2005 (7):381–90), providing further rationale for this combination study. The combination treatment was evaluated in vivo in a SCID diffuse large B-cell lymphoma model. Bortezomib treatment did not alter the CD20 expression levels of SUDHL-16 cells in vitro. MMT assays showed an additive effect of bortezomib and gamma radiation on SUDHL-16 cell proliferation. In preliminary experiments, we identified the maximal tolerated dose of ibritumomab tiuxetan in our mouse model as 50 μCi/mouse. 2x106 SUDHL-16 cells injected s.q. in the flank resulted in local tumor growth within 4 weeks. When tumors became palpable, animals were stratified in 4 different treatment groups of 12 mice each. Mice received i.v. injections of either diluent, single agent bortezomib, single agent ibritumomab tiuxetan, or the combination of both. Ibritumomab tiuxetan was injected once i.v. at a dose of 20 μCi /mouse. Bortezomib was given i.v. as single dose of 0.01 mg/mouse. The combined treatment group received i.v. bortezomib as a radio-sensitizer 3 hrs prior to the injection of ibritumomab tiuxetan. All treatment schedules delayed tumor growth compared with controls. After 16 days of treatment, bortezomib reduced tumor progression by 52%, ibritumomab tiuxetan by 69%, and the combination treatment by 91%. Selected mice were sacrificed at day two and their tumors studied for potential immediate therapeutic effects. The Y90 content of tumors and normal tissues was measured and demonstrated targeting of Y90 to the xenograft. The tumors of the control and treatment groups did not differ by microscopic morphological appearance. In situ Tunel assays did not reveal increased apoptosis at treatment day two. In summary, bortezomib as a radio-sensitizer in combination with ibritumomab tiuxetan is an active and at least additive regimen in an in vivo DLBCL model.

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The integrated stress response regulates BMP signaling through effects on translation

10.1101/266189 ◽

2018 ◽

Author(s):

Elke Malzer ◽

Caia S. Dominicus ◽

Joseph E. Chambers ◽

Souradip Mookerjee ◽

Stefan J. Marciniak

Keyword(s):

Pulmonary Hypertension ◽

Stress Response ◽

Bmp Signaling ◽

Developmental Pathways ◽

Integrated Stress Response ◽

Direct Effects ◽

Pulmonary Arterial ◽

Stress Sensing

AbstractDevelopmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Mutations of the ISR kinase GCN2 have been implicated in the development of pulmonary arterial hypertension, a disorder known to be associated with defects of BMP signaling, but how the ISR and BMP signaling might interact is unknown. Here we show in Drosophila that GCN2 antagonises BMP signaling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impair developmental BMP signaling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signaling. Moreover, loss of d4E-BP, a target of crc, augments BMP signaling in vitro and rescues tissue development in vivo. These results identify a novel mechanism by which the ISR modulates BMP signaling during development. Since abnormalities of both GCN2 and BMP signaling lead to pulmonary hypertension, these findings may have wider relevance for the development of therapies for this disease.

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HCD122, an Antagonist Human Anti-CD40 Monoclonal Antibody, Inhibits Tumor Growth in Xenograft Models of Human Diffuse Large B-Cell Lymphoma, a Subset of Non-Hodgkins Lymphoma.

Blood ◽

10.1182/blood.v108.11.2519.2519 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2519-2519 ◽

Cited By ~ 2

Author(s):

Ssucheng J. Hsu ◽

Lin A. Esposito ◽

Sharon L. Aukerman ◽

Seema Kantak ◽

Amer M. Mirza

Keyword(s):

Tumor Growth ◽

B Cell ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Non Hodgkins Lymphoma ◽

Xenograft Models

Abstract CD40, a member of the tumor necrosis factor receptor family, is expressed in all human B-cell malignancies and engagement by the CD40 ligand (CD40L) is important for both cell proliferation and cell survival. CD40L has been shown to be co-expressed with CD40 in neoplastic B-cells from Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkins Lymphoma (NHL), suggesting the importance of an autocrine CD40/CD40L loop in these malignancies. HCD122 (formerly known as CHIR-12.12) is a fully human, highly potent, IgG1 antagonist anti-CD40 monoclonal antibody (mAb) that blocks CD40/CD40L interactions in vitro and also mediates ADCC. Previous studies showed that HCD122 can mediate ADCC in vitro and has anti-proliferative and anti-tumor activities as a single agent in CLL, MM, and Burkitts Lymphoma in vitro and in vivo. In this study, the activity of HCD122 on a subtype of NHL, Diffuse Large B-Cell Lymphoma (DLBCL) was examined. The DLBCL derived cell lines, RL and SU-DHL-4, were selected for this study based upon in vivo characterization as well as their sensitivity to Rituximab as reported in the literature. These cell lines were subsequently confirmed for the expression of CD40 and CD20 by flow cytometry. The in vivo anti-tumor effects of HCD122 as single agent was demonstrated in these two xenograft models and was compared to Rituximab, an anti-CD20 antibody therapeutic currently approved for the treatment of relapsed or refractory, low-grade or follicular, NHL. HCD122 when administered intraperitoneally weekly at 1 mg/kg significantly reduced tumor growth with a tumor growth inhibition (TGI) of 85.5% (P<0.01) in the RL model. At the same dose and schedule in the RL model, TGI achieved with Rituximab was 31.7% (P>0.05). In the SU-DHL-4 model, an 85% TGI (P<0.01) was observed at the 1 mg/kg dose of HCD122. In comparison, Rituximab at this dose elicited a 57.6% TGI (P<0.05). Additionally, the downstream CD40/CD40L signal transduction pathways were also examined in order to elucidate the molecular mechanism underlying the HCD122-mediated effects in DLBCL. Taken together, these results support the clinical development of HCD122 for the treatment of DLBCL. Currently HCD122 is in Phase I trials for treatment of CLL and MM.

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Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML

Leukemia ◽

10.1038/s41375-021-01192-7 ◽

2021 ◽

Author(s):

Ilaria Gionfriddo ◽

Lorenzo Brunetti ◽

Federica Mezzasoma ◽

Francesca Milano ◽

Valeria Cardinali ◽

...

Keyword(s):

Stress Response ◽

Myeloid Leukemia ◽

Low Cost ◽

Single Agent ◽

Treatment Strategies ◽

Wild Type ◽

Nucleolar Stress ◽

Unfit Patients

AbstractAcute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.

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CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

Biochemical Journal ◽

10.1042/bcj20190320 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3141-3159 ◽

Cited By ~ 2

Author(s):

Meiru Si ◽

Can Chen ◽

Zengfan Wei ◽

Zhijin Gong ◽

GuiZhi Li ◽

...

Keyword(s):

Stress Response ◽

Ligand Binding ◽

Corynebacterium Glutamicum ◽

Conformational Changes ◽

Cysteine Oxidation ◽

Transcriptional Regulatory ◽

Ligand Free ◽

Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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