scholarly journals Gain-of-function Mutations Reveal Expanded Intermediate States and a Sequential Action of Two Gates in MscL

2005 ◽  
Vol 125 (2) ◽  
pp. 155-170 ◽  
Author(s):  
Andriy Anishkin ◽  
Chien-Sung Chiang ◽  
Sergei Sukharev
Keyword(s):  
Mechanosensitive Channel ◽  
Membrane Tension ◽  
Wild Type ◽  
Gain Of Function ◽  
Sequential Action ◽  
Intermediate States ◽  
A Minor ◽  
Open States ◽  
Rate Limiting ◽  
S States

The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) → low-conducting substate (S) → open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C→S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area (ΔA) and energy (ΔE) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger ΔE associated with the second (S→O) subtransition, but a ΔA similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (koff) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C→S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled “leaky” conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1–M1 linkers result in a much higher probability of reverse O→S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S→O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.

scholarly journals Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Griffin Chure ◽  
Heun Jin Lee ◽  
Akiko Rasmussen ◽  
Rob Phillips
Keyword(s):  
Cell Survival ◽  
Single Cell ◽  
Osmotic Shock ◽  
Single Cells ◽  
Mechanosensitive Channel ◽  
Membrane Tension ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Structural Methods

ABSTRACTRapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks,Escherichia coliand other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. InE. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in anE. colistrain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-typeE. colicells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCEMechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

scholarly journals A Gain-of-Function Mutation in Gating of Corynebacterium glutamicum NCgl1221 Causes Constitutive Glutamate Secretion

2012 ◽  
Vol 78 (15) ◽  
pp. 5432-5434 ◽  
Author(s):  
Yoshitaka Nakayama ◽  
Kenjiro Yoshimura ◽  
Hidetoshi Iida
Keyword(s):  
Patch Clamp ◽  
Corynebacterium Glutamicum ◽  
Mechanosensitive Channel ◽  
Wild Type ◽  
Gain Of Function ◽  
Content Type ◽  
Wild Type Counterpart

ABSTRACTThe A-to-V mutation at position 111 (A111V) in the mechanosensitive channel NCgl1221 (MscCG) causes constitutive glutamate secretion inCorynebacterium glutamicum. Patch clamp experiments revealed that NCgl1221 (A111V) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of NCgl1221 leads to the oversecretion of glutamate.

The Drosophila gene Bearded encodes a novel small protein and shares 3′ UTR sequence motifs with multiple Enhancer of split complex genes

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4039-4051 ◽  
Author(s):  
M.W. Leviten ◽  
E.C. Lai ◽  
J.W. Posakony
Keyword(s):  
Sequence Similarity ◽  
Notch Pathway ◽  
Alpha Helix ◽  
Sequence Motifs ◽  
Wild Type ◽  
Gain Of Function ◽  
Small Protein ◽  
Drosophila Gene ◽  
Amphipathic Alpha Helix ◽  
Enhancer Of Split

Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss phenotypes indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264–283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3′ untranslated region (UTR). The wild-type Brd 3′ UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the ‘Brd box’. Moreover, the 3′ UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the ‘GY box’ (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3′ UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic alpha-helix, suggesting that the two proteins have related biochemical functions.

Cardiac expression of a gain-of-function α5-integrin results in perinatal lethality

2001 ◽  
Vol 280 (1) ◽  
pp. H361-H367 ◽  
Author(s):  
Maria L. Valencik ◽  
John A. McDonald
Keyword(s):  
Cardiac Development ◽  
Wild Type ◽  
Integrin Receptors ◽  
Gain Of Function ◽  
Transgenic Expression ◽  
Physiological Regulation ◽  
Myocyte Differentiation ◽  
Perinatal Lethality ◽  
And Function

Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. α5β1-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of α5β1-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human α5-integrin in cardiomyocytes: the full-length wild-type α5-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated α5-1-integrin. Overexpression of the wild-type α5-integrin has no detectable adverse effects in the mouse, whereas expression of α5-1-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.

scholarly journals Mutant p53 Sequestration of the MDM2 Acidic Domain Inhibits E3 Ligase Activity

2018 ◽  
Vol 39 (4) ◽  
Author(s):  
Leixiang Yang ◽  
Tanjing Song ◽  
Qian Cheng ◽  
Lihong Chen ◽  
Jiandong Chen
Keyword(s):  
Tumor Cells ◽  
Recent Work ◽  
Intramolecular Interaction ◽  
E3 Ligase ◽  
Mutant P53 ◽  
Wild Type ◽  
Gain Of Function ◽  
Core Domain ◽  
Acidic Domain ◽  
Wild Type P53

ABSTRACT Missense p53 mutants often accumulate in tumors and drive progression through gain of function. MDM2 efficiently degrades wild-type p53 but fails to degrade mutant p53 in tumor cells. Previous studies revealed that mutant p53 inhibits MDM2 autoubiquitination, suggesting that the interaction inhibits MDM2 E3 activity. Recent work showed that MDM2 E3 activity is stimulated by intramolecular interaction between the RING and acidic domains. Here, we show that in the mutant p53-MDM2 complex, the mutant p53 core domain binds to the MDM2 acidic domain with significantly higher avidity than wild-type p53. The mutant p53-MDM2 complex is deficient in catalyzing ubiquitin release from the activated E2 conjugating enzyme. An MDM2 construct with extra copies of the acidic domain is resistant to inhibition by mutant p53 and efficiently promotes mutant p53 ubiquitination and degradation. The results suggest that mutant p53 interferes with the intramolecular autoactivation mechanism of MDM2, contributing to reduced ubiquitination and increased accumulation in tumor cells.

scholarly journals Limited effects of m6A modification on mRNA partitioning into stress granules

2021 ◽  
Author(s):  
Anthony Khong ◽  
Tyler Matheny ◽  
Thao Ngoc Huynh ◽  
Vincent Babl ◽  
Roy Parker
Keyword(s):  
Regression Analysis ◽  
Linear Regression ◽  
Single Molecule ◽  
Linear Regression Analysis ◽  
Stress Granules ◽  
Multiple Linear Regression Analysis ◽  
Minor Role ◽  
Wild Type ◽  
Specific Mrnas ◽  
A Minor

Recent studies have argued that the m6A modification of mRNAs promotes mRNA recruitment to stress granules through the interaction with YTHDF proteins (Anders et al., 2018; Ries et al., 2019). However, mRNAs that contain multiple m6A modified sites partition similarly into stress granules in both wild-type and m6A-deficient cells by single-molecule FISH suggesting m6A modifications play a minor role in mRNA partitioning into stress granules. Moreover, multiple linear regression analysis suggests m6A modification plays a minimal role in stress granule recruitment. Finally, the artificial tethering of 25 YTHDF proteins on reporter mRNAs leads to only a modest increase in mRNA partitioning to stress granules. These results indicate m6A modification makes a small, but measurable, contribution to recruiting specific mRNAs to stress granules.

Abstract 3136: Lipid-Lowering Medication Modifies The Gene-Diet Association Between 5-Lipoxygenase Promoter Polymorphisms And Carotid Artery Atherosclerosis

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Nitzan C Roth ◽  
Susanna Vikman ◽  
Hooman Allayee ◽  
Wendy J Mack ◽  
Howard N Hodis
Keyword(s):  
Carotid Artery ◽  
Intima Media Thickness ◽  
Lipid Lowering ◽  
Wild Type ◽  
Promoter Polymorphisms ◽  
Cross Sectional ◽  
Carotid Artery Atherosclerosis ◽  
Randomized Controlled ◽  
Inflammatory State ◽  
Rate Limiting

Background: 5-Lipoxygenase (5-LOX) is the rate-limiting enzyme in the biosynthesis of leukotrienes from arachidonic acid (Ara), an n-6 polyunsaturated fatty acid (PUFA). Previous studies suggest that polymorphisms in the number of tandem Sp1 binding sites in the promoter of the 5-LOX gene may have an atherogenic effect due to a heightened inflammatory state when dietary Ara is high and dietary long-chain n-3 PUFAs (eicosapentaenoic acid, EPA, plus docosahexaenoic acid, DHA) are low. Methods: We determined 5-LOX genotypes in 1,663 adults participating in one of five randomized controlled atherosclerosis trials using carotid artery intima-media thickness (CIMT) as the outcome. Baseline data on participant characteristics and clinic and laboratory measurements, including fasting lipids and CIMT, were obtained using the same methods in all trials. Diet was measured using 3-day records and intake of each PUFA was categorized as above or below the median. We used linear regression to examine the cross-sectional associations between 5-LOX genotype, dietary PUFAs, and CIMT. Results: Alleles were classified as wild-type (W, with 5 repeats), deletion (D, with 2– 4 repeats), or addition (A, with 6–9 repeats). The frequencies of the six genotypes were: 60.6% WW, 27.1% WD, 4.9% WA, 4.9% DD, 2.2% DA, and 0.4% AA. Among participants using lipid-lowering medication, mean CIMT was significantly elevated by 79.1 μm (95% CI = 5.3–152.9) in DD individuals compared to WW individuals after adjustment for sex, race, age, trial, and BMI >25 kg/m 2 . High dietary EPA+DHA blunted the gene-CIMT association (p for interaction = 0.007). In contrast, DD genotype was not associated with CIMT among non-users of lipid-lowering medication. Conclusion: The atherogenic effect of the shorter promoter alleles is modulated by dietary intake, especially in individuals using lipid-lowering medication, indicating a complex mechanism involving both inflammation and lipids.

Characterization of Holliday structures in FLP protein-promoted site-specific recombination

1990 ◽  
Vol 10 (1) ◽  
pp. 235-242
Author(s):  
L Meyer-Leon ◽  
R B Inman ◽  
M M Cox
Keyword(s):  
Reaction Rate ◽  
Reaction Pathway ◽  
Reaction Intermediates ◽  
Wild Type ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Isomerization Process ◽  
Previous Demonstration ◽  
Rate Limiting

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

scholarly journals Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis

2020 ◽  
Vol 21 (2) ◽  
pp. 644 ◽  
Author(s):  
Eva B. Znalesniak ◽  
Franz Salm ◽  
Werner Hoffmann
Keyword(s):  
Cysteine Residue ◽  
Molecular Forms ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Rt Pcr ◽  
Molecular Alterations ◽  
Protein Levels ◽  
Increased Sensitivity ◽  
A Minor ◽  
Deficient Mice

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

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