scholarly journals Multiple Myeloma Cells Depend on the DDI2/NRF1-mediated Proteasome Stress Response for Survival

2021 ◽  
Author(s):  
Tianzeng Chen ◽  
Matthew Ho ◽  
Jenna Briere ◽  
Maria Moscvin ◽  
Peter G Czarnecki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stress Response ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
De Novo ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Knock Out

Multiple myeloma (MM) cells suffer from baseline proteotoxicity due to an imbalance between the load of misfolded proteins awaiting proteolysis and the capacity of the ubiquitin-proteasome system to degrade them. This intrinsic vulnerability is at the base of MM sensitivity to agents that perturb proteostasis such as proteasome inhibitors (PIs), the mainstay of modern-day myeloma therapy. De-novo and acquired PI resistance are important clinical limitations, adversely affecting prognosis. The molecular mechanisms underpinning PI resistance are only partially understood, limiting the development of drugs that can overcome it. The transcription factor NRF1 is activated by the aspartic protease DDI2 upon proteasome insufficiency and governs proteasome biogenesis. In this work, we show that MM cells exhibit baseline NRF1 activation and are dependent upon DDI2 for survival. DDI2 knock out (KO) is cytotoxic for MM cells, both in vitro and in vivo. Protein structure-function studies show that DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome activity recovery upon irreversible proteasome inhibition, thereby increasing sensitivity to PI. Add-back of wild-type, but not of catalytically-dead DDI2, fully rescues these phenotypes. We propose that DDI2 is an unexplored, promising molecular target in MM by disrupting the proteasome stress response and exacerbating proteotoxicity.

scholarly journals Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi

2016 ◽  
Vol 84 (11) ◽  
pp. 3141-3151 ◽  
Author(s):  
Adrienne C. Showman ◽  
George Aranjuez ◽  
Philip P. Adams ◽  
Mollie W. Jewett
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Borrelia Burgdorferi ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Oxidative Stress Response ◽  
Growth Defect ◽  
Content Type

A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo , we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δ bb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 . In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.

Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form

1991 ◽  
Vol 11 (1) ◽  
pp. 401-411
Author(s):  
S Cuthill ◽  
A Wilhelmsson ◽  
L Poellinger
Keyword(s):  
Dna Binding ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Rank Order ◽  
Receptor Ligands ◽  
Free System ◽  
Dioxin Receptor

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

scholarly journals Glycosylation of CaV3.2 Channels Contributes to the Hyperalgesia in Peripheral Neuropathy of Type 1 Diabetes

2020 ◽  
Vol 14 ◽  
Author(s):  
Sonja Lj. Joksimovic ◽  
J. Grayson Evans ◽  
William E. McIntire ◽  
Peihan Orestes ◽  
Paula Q. Barrett ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Adult Female ◽  
Molecular Mechanisms ◽  
Sprague Dawley ◽  
Knock Out ◽  
Peripheral Diabetic Neuropathy ◽  
Novel Treatments

Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.

scholarly journals Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1654-1664 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita Singh ◽  
Mohan Brahmandam ◽  
Klaus Podar ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Er Stress Response ◽  
Synergistic Cytotoxicity ◽  
Proteolytic Activities ◽  
Dose Combination ◽  
Inhibition Of Tumor Growth

AbstractOur recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib–induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-κB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib–treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid–based proteasome inhibitors

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 5927-5937 ◽  
Author(s):  
Encouse B. Golden ◽  
Philip Y. Lam ◽  
Adel Kardosh ◽  
Kevin J. Gaffney ◽  
Enrique Cadenas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Tumor Cell ◽  
Green Tea ◽  
Boronic Acid ◽  
Proteasome Inhibitors ◽  
Green Tea Polyphenols ◽  
Tumor Cell Death

Abstract The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this “miracle herb” in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid–based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non–boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

scholarly journals TIE2 Associates with Caveolae and Regulates Caveolin-1 To Promote Their Nuclear Translocation

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Mohammad B. Hossain ◽  
Rehnuma Shifat ◽  
Jingyi Li ◽  
Xuemei Luo ◽  
Kenneth R. Hess ◽  
...  
Keyword(s):  
Dna Repair ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Subcellular Fractionation ◽  
Tyrosine Kinase Receptor ◽  
Histone H4 ◽  
Small Interfering Rnas ◽  
Caveolin 1

ABSTRACT DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies—chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 in vitro and in vivo. This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

scholarly journals Modulating proteasome inhibitor tolerance in multiple myeloma: an alternative strategy to reverse inevitable resistance

2020 ◽  
Author(s):  
Maolin Ge ◽  
Zhi Qiao ◽  
Yan Kong ◽  
Hongyu Liang ◽  
Yan Sun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Histone Deacetylase Inhibitors ◽  
Resistance Mechanism ◽  
Proteasome Inhibitors ◽  
Intermittent Therapy ◽  
Bioinformatics Analyses ◽  
Inhibitor Tolerance ◽  
Epigenetic Regulators

Abstract Background Resistance to proteasome inhibitors (PIs) is a major obstacle to the successful treatment of multiple myeloma (MM). Many mechanisms have been proposed for PI resistance; however, our mechanistic understanding of how PI resistance is inevitably acquired and reversed remains incomplete. Methods MM patients after bortezomib relapse, MM cell lines and mouse models were used to generate matched resistant and reversed cells. RNA sequencing and bioinformatics analyses were employed to assess dysregulated epigenetic regulators. In vitro and in vivo procedures were used to characterise PI-tolerant cells and therapeutic efficacy. Results Upon PI treatment, MM cells enter a slow-cycling and reversible drug-tolerant state. This reversible phenotype is associated with epigenetic plasticity, which involves tolerance rather than persistence in patients with relapsed MM. Combination treatment with histone deacetylase inhibitors and high-dosage intermittent therapy, as opposed to sustained PI monotherapy, can be more effective in treating MM by preventing the emergence of PI-tolerant cells. The therapeutic basis is the reversal of dysregulated epigenetic regulators in MM patients. Conclusions We propose an alternative non-mutational PI resistance mechanism that explains why PI relapse is inevitable and why patients regain sensitivity after a ‘drug holiday’. Our study also suggests strategies for epigenetic elimination of drug-tolerant cells.

scholarly journals CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi47
Author(s):  
Marilin Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Justyna Przystal ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Profile ◽  
Transcriptional Networks ◽  
Molecular Alterations ◽  
Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

scholarly journals Insight into Nephrocan Function in Mouse Endoderm Patterning

2019 ◽  
Vol 21 (1) ◽  
pp. 8
Author(s):  
Martina Addeo ◽  
Silvia Buonaiuto ◽  
Ilaria Guerriero ◽  
Elena Amendola ◽  
Feliciano Visconte ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Specific Expression ◽  
Knock Out ◽  
New Variant ◽  
Liver And Pancreas ◽  
Regenerative Therapies

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5–11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn−/− mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

In vitro reconstitution of autophagic processes

2020 ◽  
Vol 48 (5) ◽  
pp. 2003-2014
Author(s):  
Jahangir Md. Alam ◽  
Nobuo N. Noda
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
The Past ◽  
Atg Proteins ◽  
In Vitro Reconstitution ◽  
Complicated Process

Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations.

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