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.

Novel Findings of Anti-cancer Property of Propofol

2018 ◽  
Vol 18 (2) ◽  
pp. 156-165 ◽  
Author(s):  
Jiaqiang Wang ◽  
Chien-shan Cheng ◽  
Yan Lu ◽  
Xiaowei Ding ◽  
Minmin Zhu ◽  
...  
Keyword(s):  
General Anesthesia ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Intravenous Anesthetic ◽  
The Past ◽  
Anti Cancer ◽  
Underlying Mechanisms ◽  
Recent Attention

Background: Propofol, a widely used intravenous anesthetic agent, is traditionally applied for sedation and general anesthesia. Explanation: Recent attention has been drawn to explore the effect and mechanisms of propofol against cancer progression in vitro and in vivo. Specifically, the proliferation-inhibiting and apoptosis-inducing properties of propofol in cancer have been studied. However, the underlying mechanisms remain unclear. Conclusion: This review focused on the findings within the past ten years and aimed to provide a general overview of propofol's malignance-modulating properties and the potential molecular mechanisms.

scholarly journals Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy

2014 ◽  
Vol 37 (6) ◽  
pp. E12 ◽  
Author(s):  
Encouse B. Golden ◽  
Hee-Yeon Cho ◽  
Ardeshir Jahanian ◽  
Florence M. Hofman ◽  
Stan G. Louie ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
In Vivo Model ◽  
Autophagosome Formation ◽  
Individual Drug ◽  
In Vivo Experiments ◽  
Associated Proteins

Object In a recent clinical trial, patients with newly diagnosed glioblastoma multiforme benefited from chloroquine (CQ) in combination with conventional therapy (resection, temozolomide [TMZ], and radiation therapy). In the present study, the authors report the mechanism by which CQ enhances the therapeutic efficacy of TMZ to aid future studies aimed at improving this therapeutic regimen. Methods Using in vitro and in vivo experiments, the authors determined the mechanism by which CQ enhances TMZ cytotoxicity. They focused on the inhibition-of-autophagy mechanism of CQ by knockdown of the autophagy-associated proteins or treatment with autophagy inhibitors. This mechanism was tested using an in vivo model with subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ. Results Knockdown of the autophagy-associated proteins (GRP78 and Beclin) or treatment with the autophagy inhibitor, 3-methyl adenine (3-MA), blocked autophagosome formation and reduced CQ cytotoxicity, suggesting that autophagosome accumulation precedes CQ-induced cell death. In contrast, blocking autophagosome formation with knockdown of GRP78 or treatment with 3-MA enhanced TMZ cytotoxicity, suggesting that the autophagy pathway protects from TMZ-induced cytotoxicity. CQ in combination with TMZ significantly increased the amounts of LC3B-II (a marker for autophagosome levels), CHOP/GADD-153, and cleaved PARP (a marker for apoptosis) over those with untreated or individual drug-treated glioma cells. These molecular mechanisms seemed to take place in vivo as well. Subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ displayed higher levels of CHOP/GADD-153 than did untreated or individual drug-treated tumors. Conclusions Taken together, these results demonstrate that CQ blocks autophagy and triggers endoplasmic reticulum stress, thereby increasing the chemosensitivity of glioma cells to TMZ.

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence

1998 ◽  
Vol 111 (6) ◽  
pp. 759-767
Author(s):  
G. Herrmann ◽  
P. Brenneisen ◽  
M. Wlaschek ◽  
J. Wenk ◽  
K. Faisst ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Molecular Data ◽  
Cellular Aging ◽  
Premature Aging ◽  
Functional Changes ◽  
Uva Irradiation ◽  
High Level

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of >120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

scholarly journals Endothelial Sphingolipid De Novo Synthesis Controls Blood Pressure by Regulating Signal Transduction and NO via Ceramide

Hypertension ◽  
2020 ◽  
Vol 75 (5) ◽  
pp. 1279-1288 ◽  
Author(s):  
Anna Cantalupo ◽  
Linda Sasset ◽  
Antonella Gargiulo ◽  
Luisa Rubinelli ◽  
Ilaria Del Gaudio ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Signal Transduction ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Control Flow ◽  
Serine Palmitoyltransferase ◽  
Blood Pressure Homeostasis

Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus–related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)—the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.

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.

scholarly journals Oxidative and Electrophilic Stresses Activate Nrf2 through Inhibition of Ubiquitination Activity of Keap1

2006 ◽  
Vol 26 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Akira Kobayashi ◽  
Moon-Il Kang ◽  
Yoriko Watai ◽  
Kit I. Tong ◽  
Takahiro Shibata ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Degradation Mechanism ◽  
Nuclear Accumulation ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Nrf2 Activation ◽  
Nrf2 Protein

ABSTRACT The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

scholarly journals Curvature-sensitive trans-assembly of human Atg8-family proteins in autophagy-related membrane tethering

2019 ◽  
Author(s):  
Saki Taniguchi ◽  
Masayuki Toyoshima ◽  
Tomoyo Takamatsu ◽  
Joji Mima
Keyword(s):  
Lipid Membranes ◽  
De Novo ◽  
Membrane Curvature ◽  
Membrane Dynamics ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Molar Ratios ◽  
Membrane Tethering ◽  
Membrane Attachment

In macroautophagy, de novo formation of the double membrane-bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8-family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8-mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE-16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE-16 retained the capacities to trigger efficient membrane tethering at the protein-to-lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8-mediated membrane tethering reactions require trans-assembly between the membrane-anchored forms of LC3B and GATE-16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B- and GATE-16-mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE-16 for highly-curved small vesicles (e.g. 50 nm in diameter), although GATE-16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g. 200 and 400 nm in diameter). Our findings establish curvature-sensitive trans-assembly of human Atg8-family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.

scholarly journals Expression Levels of Glycoprotein O (gO) Vary between Strains of Human Cytomegalovirus, Influencing the Assembly of gH/gL Complexes and Virion Infectivity

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Le Zhang ◽  
Momei Zhou ◽  
Richard Stanton ◽  
Jeremy Kamil ◽  
Brent J. Ryckman
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Purifying Selection ◽  
Selective Advantage ◽  
Immune Recognition ◽  
Expression Levels

ABSTRACT The tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the endoplasmic reticulum (ER). This assembly process clearly differs among strains, since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much lower levels of gH/gL/UL128-131 than ME. Remaining questions include (i) what are the mechanisms behind these assembly differences, and (ii) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) open reading frame (ORF) differs in 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced the infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression levels were within 4-fold between TR and ME, but the gO expression level was 20-fold lower for ME, which suggested differences in mRNA transcription, translation, or rapid ER-associated degradation of gO. trans-Complementation of gO expression during ME replication gave a 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in the assembly of gH/gL complexes result from differences in the expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than those for higher gO expression. IMPORTANCE Specific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo. This may have important implications for virus-host interactions, such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

scholarly journals Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity

2018 ◽  
Author(s):  
Le Zhang ◽  
Momei Zhou ◽  
Richard Stanton ◽  
Jeremy Kamil ◽  
Brent J. Ryckman
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Purifying Selection ◽  
Selective Advantage ◽  
Immune Recognition ◽  
Virion Assembly

ABSTRACTTropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the ER. This assembly process clearly differs among strains since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much less gH/gL/UL128-131 than ME. Remaining questions include 1) what are the mechanisms behind these assembly differences, and 2) do differences reflectin vitroculture adaptations or natural genetic variations? Since the UL74(gO) ORF differs by 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA transcription, translation or rapid ER-associated degradation of gO. Trans-complementation of gO expression during ME replication gave 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in assembly of gH/gL complexes result from differences in expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than for higher gO expression.IMPORTANCESpecific genetic differences between independently isolated HCMV strains may result from purifying selection onde novomutations arising during propagation in culture, or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability presentin vivo. This may have important implications for virus-host interactions such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

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.

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