scholarly journals The step-wise pathway of septin hetero-octamer assembly in budding yeast

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Andrew Weems ◽  
Michael McMurray
Keyword(s):  
Budding Yeast ◽  
Underlying Mechanism ◽  
Guanine Nucleotides ◽  
Gtp Hydrolysis ◽  
The Core ◽  
Assembly Pathway ◽  
N Terminus ◽  
Specific Affinity ◽  
Stepwise Assembly

Septin proteins bind guanine nucleotides and form rod-shaped hetero-oligomers. Cells choose from a variety of available septins to assemble distinct hetero-oligomers, but the underlying mechanism was unknown. Using a new in vivo assay, we find that a stepwise assembly pathway produces the two species of budding yeast septin hetero-octamers: Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Rapid GTP hydrolysis by monomeric Cdc10 drives assembly of the core Cdc10 homodimer. The extended Cdc3 N terminus autoinhibits Cdc3 association with Cdc10 homodimers until prior Cdc3–Cdc12 interaction. Slow hydrolysis by monomeric Cdc12 and specific affinity of Cdc11 for transient Cdc12•GTP drive assembly of distinct trimers, Cdc11–Cdc12–Cdc3 or Shs1–Cdc12–Cdc3. Decreasing the cytosolic GTP:GDP ratio increases the incorporation of Shs1 vs Cdc11, which alters the curvature of filamentous septin rings. Our findings explain how GTP hydrolysis controls septin assembly, and uncover mechanisms by which cells construct defined septin complexes.

scholarly journals Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Vol 117 (45) ◽  
pp. 27989-27996
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Wild Type ◽  
Protease Domain ◽  
Loop Region ◽  
Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

scholarly journals Reversible auto-inhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Loop Region ◽  
Assembly Pathway ◽  
Inhibitory Regulation

AbstractEscherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD, but the underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features, in particular the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has a potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of the BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of the protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Tele-Substitution Reactions in the Synthesis of a Promising Class of Antimalarials

2020 ◽  
Author(s):  
Marat Korsik ◽  
Edwin Tse ◽  
David Smith ◽  
William Lewis ◽  
Peter J. Rutledge ◽  
...  
Keyword(s):  
Heterocyclic Ring ◽  
Ring System ◽  
Substitution Reactions ◽  
Laboratory Notebook ◽  
Polar Solvents ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Core ◽  
Nmr Data

<p></p><p>We have discovered and studied a <i>tele</i>substitution reaction in a biologically important heterocyclic ring system. Conditions that favour the <i>tele</i>-substitution pathway were identified: the use of increased equivalents of the nucleophile or decreased equivalents of base, or the use of softer nucleophiles, less polar solvents and larger halogens on the electrophile. Using results from X-ray crystallography and isotope labelling experiments a mechanism for this unusual transformation is proposed. We focused on this triazolopyrazine as it is the core structure of the <i>in vivo </i>active anti-plasmodium compounds of Series 4 of the Open Source Malaria consortium.</p> <p> </p> <p>Archive of the electronic laboratory notebook with the description of all conducted experiments and raw NMR data could be accessed via following link <a href="https://ses.library.usyd.edu.au/handle/2123/21890">https://ses.library.usyd.edu.au/handle/2123/21890</a> . For navigation between entries of laboratory notebook please use file "Strings for compounds in the article.pdf" that works as a reference between article codes and notebook codes, also this file contain SMILES for these compounds. </p><br><p></p>

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals GCH1 induces immunosuppression through metabolic reprogramming and IDO1 upregulation in triple-negative breast cancer

2021 ◽  
Vol 9 (7) ◽  
pp. e002383
Author(s):  
Jin-Li Wei ◽  
Si-Yu Wu ◽  
Yun-Song Yang ◽  
Yi Xiao ◽  
Xi Jin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Molecular Taxonomy ◽  
Underlying Mechanism ◽  
Metabolic Reprogramming ◽  
Sequencing Data ◽  
Aryl Hydrocarbon

PurposeRegulatory T cells (Tregs) heavily infiltrate triple-negative breast cancer (TNBC), and their accumulation is affected by the metabolic reprogramming in cancer cells. In the present study, we sought to identify cancer cell-intrinsic metabolic modulators correlating with Tregs infiltration in TNBC.Experimental designUsing the RNA-sequencing data from our institute (n=360) and the Molecular Taxonomy of Breast Cancer International Consortium TNBC cohort (n=320), we calculated the abundance of Tregs in each sample and evaluated the correlation between gene expression levels and Tregs infiltration. Then, in vivo and in vitro experiments were performed to verify the correlation and explore the underlying mechanism.ResultsWe revealed that GTP cyclohydrolase 1 (GCH1) expression was positively correlated with Tregs infiltration and high GCH1 expression was associated with reduced overall survival in TNBC. In vivo and in vitro experiments showed that GCH1 increased Tregs infiltration, decreased apoptosis, and elevated the programmed cell death-1 (PD-1)-positive fraction. Metabolomics analysis indicated that GCH1 overexpression reprogrammed tryptophan metabolism, resulting in L-5-hydroxytryptophan (5-HTP) accumulation in the cytoplasm accompanied by kynurenine accumulation and tryptophan reduction in the supernatant. Subsequently, aryl hydrocarbon receptor, activated by 5-HTP, bound to the promoter of indoleamine 2,3-dioxygenase 1 (IDO1) and thus enhanced the transcription of IDO1. Furthermore, the inhibition of GCH1 by 2,4-diamino-6-hydroxypyrimidine (DAHP) decreased IDO1 expression, attenuated tumor growth, and enhanced the tumor response to PD-1 blockade immunotherapy.ConclusionsTumor-cell-intrinsic GCH1 induced immunosuppression through metabolic reprogramming and IDO1 upregulation in TNBC. Inhibition of GCH1 by DAHP serves as a potential immunometabolic strategy in TNBC.

scholarly journals Development of a potent embryonic chick lens model for studying congenital cataracts in vivo

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Zhen Li ◽  
Sumin Gu ◽  
Yumeng Quan ◽  
Kulandaiappan Varadaraj ◽  
Jean X. Jiang
Keyword(s):  
Light Transmission ◽  
Genetic Diseases ◽  
Gene Mutations ◽  
Underlying Mechanism ◽  
Dominant Negative ◽  
Lens Model ◽  
Embryonic Chick ◽  
Congenital Cataracts ◽  
Chick Lens

AbstractCongenital cataracts are associated with gene mutations, yet the underlying mechanism remains largely unknown. Here we reported an embryonic chick lens model that closely recapitulates the process of cataract formation. We adopted dominant-negative site mutations that cause congenital cataracts, connexin, Cx50E48K, aquaporin 0, AQP0R33C, αA-crystallin, CRYAA R12C and R54C. The recombinant retroviruses containing these mutants were microinjected into the occlusive lumen of chick lenses at early embryonic development. Cx50E48K expression developed cataracts associated with disorganized nuclei and enlarged extracellular spaces. Expression of AQP0R33C resulted in cortical cataracts, enlarged extracellular spaces and distorted fiber cell organization. αA crystallin mutations distorted lens light transmission and increased crystalline protein aggregation. Together, retroviral expression of congenital mutant genes in embryonic chick lenses closely mimics characteristics of human congenital cataracts. This model will provide an effective, reliable in vivo system to investigate the development and underlying mechanism of cataracts and other genetic diseases.

HG-9-91-01 Attenuates Murine Experimental Colitis by Promoting Interleukin-10 Production in Colonic Macrophages Through the SIK/CRTC3 Pathway

2021 ◽  
Author(s):  
Yong Fu ◽  
Gailing Ma ◽  
Yuqian Zhang ◽  
Wenli Wang ◽  
Tongguo Shi ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Experimental Colitis ◽  
Underlying Mechanism ◽  
Interleukin 10 ◽  
Camp Response Element Binding ◽  
Cellular Level ◽  
Trinitrobenzene Sulfonic Acid ◽  
Murine Colitis ◽  
Anti Inflammatory

Abstract Background Interleukin-10 (IL-10) is a potent immunoregulatory cytokine that plays a pivotal role in maintaining mucosal immune homeostasis. As a novel synthetic inhibitor of salt-inducible kinases (SIKs), HG-9-91-01 can effectively enhance IL-10 secretion at the cellular level, but its in vivo immunoregulatory effects remain unclear. In this study, we investigated the effects and underlying mechanism of HG-9-91-01 in murine colitis models. Methods The anti-inflammatory effects of HG-9-91-01 were evaluated on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-, dextran sulfate sodium–induced colitis mice, and IL-10 knockout chronic colitis mice. The in vivo effector cell of HG-9-91-01 was identified by fluorescence-activated cell sorting and quantitative real-time polymerase chain reaction. The underlying mechanism of HG-9-91-01 was investigated via overexpressing SIKs in ANA-1 macrophages and TNBS colitis mice. Results Treatment with HG-9-91-01 showed favorable anticolitis effects in both TNBS- and DSS-treated mice through significantly promoting IL-10 expression in colonic macrophages but failed to protect against IL-10 KO murine colitis. Further study indicated that HG-9-91-01 markedly enhanced the nuclear level of cAMP response element-binding protein (CREB)-regulated transcription coactivator 3 (CRTC3), whereas treatment with lentiviruses encoding SIK protein markedly decreased the nuclear CRTC3 level in HG-9-91-01–treated ANA-1 macrophages. In addition, intracolonic administration with lentiviruses encoding SIK protein significantly decreased the nuclear CRTC3 level in the lamina propria mononuclear cells and ended the anti-inflammatory activities of HG-9-91-01. Conclusions We found that HG-9-91-01 promoted the IL-10 expression of colonic macrophages and exhibited its anticolitis activity through the SIK/CRTC3 axis, and thus it may represent a promising strategy for inflammatory bowel disease therapy.

scholarly journals Cancer-associated fibroblasts promote the survival of irradiated nasopharyngeal carcinoma cells via the NF-κB pathway

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Weiqiang Huang ◽  
Longshan Zhang ◽  
Mi Yang ◽  
Xixi Wu ◽  
Xiaoqing Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nasopharyngeal Carcinoma ◽  
Enrichment Analysis ◽  
Underlying Mechanism ◽  
Gene Set Enrichment Analysis ◽  
Cancer Associated Fibroblasts ◽  
Western Blot Assay ◽  
Damage Level ◽  
Radiation Resistant

Abstract Background Irradiation has emerged as a valid tool for nasopharyngeal carcinoma (NPC) in situ treatment; however, NPC derived from tissues treated with irradiation is a main cause cancer-related death. The purpose of this study is to uncover the underlying mechanism regarding tumor growth after irradiation and provided potential therapeutic strategy. Methods Fibroblasts were extracted from fresh NPC tissue and normal nasopharyngeal mucosa. Immunohistochemistry was conducted to measure the expression of α-SMA and FAP. Cytokines were detected by protein array chip and identified by real-time PCR. CCK-8 assay was used to detect cell proliferation. Radiation-resistant (IRR) 5-8F cell line was established and colony assay was performed to evaluate tumor cell growth after irradiation. Signaling pathways were acquired via gene set enrichment analysis (GSEA). Comet assay and γ-H2AX foci assay were used to measure DNA damage level. Protein expression was detected by western blot assay. In vivo experiment was performed subcutaneously. Results We found that radiation-resistant NPC tissues were constantly infiltrated with a greater number of cancer-associated fibroblasts (CAFs) compared to radiosensitive NPC tissues. Further research revealed that CAFs induced the formation of radioresistance and promoted NPC cell survival following irradiation via the IL-8/NF-κB pathway to reduce irradiation-induced DNA damage. Treatment with Tranilast, a CAF inhibitor, restricted the survival of CAF-induced NPC cells and attenuated the of radioresistance properties. Conclusions Together, these data demonstrate that CAFs can promote the survival of irradiated NPC cells via the NF-κB pathway and induce radioresistance that can be interrupted by Tranilast, suggesting the potential value of Tranilast in sensitizing NPC cells to irradiation.

scholarly journals A novel hypoxic long noncoding RNA KB-1980E6.3 maintains breast cancer stem cell stemness via interacting with IGF2BP1 to facilitate c-Myc mRNA stability

Oncogene ◽  
2021 ◽  
Author(s):  
Pengpeng Zhu ◽  
Fang He ◽  
Yixuan Hou ◽  
Gang Tu ◽  
Qiao Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Underlying Mechanism ◽  
Rapid Expansion ◽  
Breast Cancer Patients ◽  
Coding Region ◽  
Signaling Axis

AbstractThe hostile hypoxic microenvironment takes primary responsibility for the rapid expansion of breast cancer tumors. However, the underlying mechanism is not fully understood. Here, using RNA sequencing (RNA-seq) analysis, we identified a hypoxia-induced long noncoding RNA (lncRNA) KB-1980E6.3, which is aberrantly upregulated in clinical breast cancer tissues and closely correlated with poor prognosis of breast cancer patients. The enhanced lncRNA KB-1980E6.3 facilitates breast cancer stem cells (BCSCs) self-renewal and tumorigenesis under hypoxic microenvironment both in vitro and in vivo. Mechanistically, lncRNA KB-1980E6.3 recruited insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to form a lncRNA KB-1980E6.3/IGF2BP1/c-Myc signaling axis that retained the stability of c-Myc mRNA through increasing binding of IGF2BP1 with m6A-modified c-Myc coding region instability determinant (CRD) mRNA. In conclusion, we confirm that lncRNA KB-1980E6.3 maintains the stemness of BCSCs through lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis and suggest that disrupting this axis might provide a new therapeutic target for refractory hypoxic tumors.

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