scholarly journals The Mechanical Power of Protein Folding

2018 ◽  
Author(s):  
Edward C. Eckels ◽  
Shubhasis Haldar ◽  
Rafael Tapia-Rojo ◽  
Jaime Andres Rivas Pardo ◽  
Julio M. Fernández
Keyword(s):  
Protein Folding ◽  
Functional Significance ◽  
Disulfide Bonds ◽  
Tandem Repeats ◽  
Muscle Protein ◽  
Muscle Mechanics ◽  
Magnetic Tweezers ◽  
Mechanical Power ◽  
Ig Domain

AbstractThe delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between force and velocity of its constituent molecular systems. Here we demonstrate a switchable power amplifier in an Ig domain of the massive muscle protein titin. Titin is composed of many tandem repeats of individually foldable Ig domains, which unfold and extend during muscle stretch and readily refold when the force on titin is quenched during a contraction. Cryptic cysteine residues are common in elastic proteins like titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding domain. However, the functional significance of disulfide-bonds in titin Ig domains remains unknown and may be fundamental to muscle mechanics. Here we use ultra-stable magnetic tweezers force spectroscopy to study the elasticity of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time using both organic reagents and oxidoreductase enzymes. We show that presence of an oxidized disulfide bond allows the parent Ig domain to fold at much higher forces, shifting the midpoint folding probability from 4.0 pN to 12.8 pN after formation. The presence of disulfide bonds in titin regulates the power output of protein folding in an all-or-none manner, providing for example at 6.0 pN, a boost from 0 to 6,000 zeptowatts upon oxidation. At this same force, single molecular motors such as myosin are typically stalled and perform little to no work. We further demonstrate that protein disulfide isomerase (PDI) readily reintroduces disulfide bonds into unfolded titin Ig domains, an important mechanism for titin which operates under a resting force of several pNin vivo. Our results demonstrate, for the first time, the functional significance of disulfide bonds as potent power amplifiers in titin and provide evidence that protein folding can generate substantial amounts of power to supplement the myosin motors during a contraction.

Abstract 2: MANF, A Structurally Unique Redox-Sensitive Chaperone, Restores ER Protein Folding in the Ischemic Heart.

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Adrian Arrieta ◽  
Erik A Blackwood ◽  
Winston T Stauffer ◽  
Michelle Santo Domingo ◽  
Amber N Pentoney ◽  
...  
Keyword(s):  
Protein Folding ◽  
Er Stress ◽  
Disulfide Bonds ◽  
Redox Status ◽  
Ischemic Heart ◽  
Neonatal Rat ◽  
Misfolded Proteins ◽  
Loss Of Function

Rationale: In cardiomyocytes, most secreted and membrane proteins are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that during myocardial ischemia, decreased oxygen creates a reducing environment in the SR/ER, preventing protein disulfide isomerases (PDIs) from forming disulfide bonds in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins which contributes to cardiomyocyte death. In response to ER stress, the transcription factor, ATF6 induces chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function cannot be inferred from other proteins. Since MANF is induced by ATF6, is ER-localized, and contains a conserved redox-sensitive motif found in PDIs, we hypothesized that MANF is a redox-sensitive chaperone that optimizes cardiomyocyte viability during ischemia. Methods: The redox status of MANF during reductive ER stress and the ability of MANF to bind misfolded proteins during ischemia were assessed in neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to suppress aggregation of misfolded proteins was examined in an in vitro chaperone assay. Finally, the effects of MANF loss-of-function in the ischemic heart, in vivo , were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA. Results: In NRVM subjected to ER stress MANF was as sensitive to changes in ER redox status as the sentinel PDI, PDIA1. Moreover, MANF formed disulfide-linked complexes with misfolded proteins during ischemia-mediated ER stress. Under reducing conditions, recombinant MANF suppressed aggregation of model misfolded proteins, in vitro . MANF knockdown in the heart, in vivo , increased damage from myocardial infarction, and an AAV9-based gene therapy approach rescued the effects of MANF deficiency, in vivo. Conclusions: MANF is a redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and decreases tissue damage in the ischemic heart.

scholarly journals Analysis of the Isomerase and Chaperone-Like Activities of an Amebic PDI (EhPDI)

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Rosa E. Mares ◽  
Alexis Z. Minchaca ◽  
Salvador Villagrana ◽  
Samuel G. Meléndez-López ◽  
Marco A. Ramos
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Molecular Mechanisms ◽  
Thermal Inactivation ◽  
Initial Step ◽  
E Coli ◽  
Isomerase Activity ◽  
Disulfide Isomerases ◽  
Protein Disulfide

Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. TheEhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation inE. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant ofE. coli, to test whetherEhPDI exhibits isomerase activityin vivo. Our preliminary results showed thatEhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed thatEhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization ofEhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding inE. histolytica.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

scholarly journals Changes in electromyographic activity, mechanical power, and relaxation rates following inspiratory ribcage muscle fatigue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Antonio Sarmento ◽  
Guilherme Fregonezi ◽  
Maria Lira ◽  
Layana Marques ◽  
Francesca Pennati ◽  
...  
Keyword(s):  
Muscle Fatigue ◽  
Low Frequency ◽  
Mechanical Power ◽  
Electromyographic Activity ◽  
Median Frequency ◽  
Relaxation Rates ◽  
Shortening Velocity ◽  
Inspiratory Muscles ◽  
Underlying Mechanisms

AbstractMuscle fatigue is a complex phenomenon enclosing various mechanisms. Despite technological advances, these mechanisms are still not fully understood in vivo. Here, simultaneous measurements of pressure, volume, and ribcage inspiratory muscle activity were performed non-invasively during fatigue (inspiratory threshold valve set at 70% of maximal inspiratory pressure) and recovery to verify if inspiratory ribcage muscle fatigue (1) leads to slowing of contraction and relaxation properties of ribcage muscles and (2) alters median frequency and high-to-low frequency ratio (H/L). During the fatigue protocol, sternocleidomastoid showed the fastest decrease in median frequency and slowest decrease in H/L. Fatigue was also characterized by a reduction in the relative power of the high-frequency and increase of the low-frequency. During recovery, changes in mechanical power were due to changes in shortening velocity with long-lasting reduction in pressure generation, and slowing of relaxation [i.e., tau (τ), half-relaxation time (½RT), and maximum relaxation rate (MRR)] was observed with no significant changes in contractile properties. Recovery of median frequency was faster than H/L, and relaxation rates correlated with shortening velocity and mechanical power of inspiratory ribcage muscles; however, with different time courses. Time constant of the inspiratory ribcage muscles during fatigue and recovery is not uniform (i.e., different inspiratory muscles may have different underlying mechanisms of fatigue), and MRR, ½RT, and τ are not only useful predictors of inspiratory ribcage muscle recovery but may also share common underlying mechanisms with shortening velocity.

scholarly journals Predicting Absorption-Distribution Properties of Neuroprotective Phosphine-Borane Compounds Using In Silico Modeling and Machine Learning

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2505
Author(s):  
Raheem Remtulla ◽  
Sanjoy Kumar Das ◽  
Leonard A. Levin
Keyword(s):  
Machine Learning ◽  
Neural Networks ◽  
In Silico ◽  
Disulfide Bonds ◽  
Oral Absorption ◽  
In Vivo Studies ◽  
Drug Candidates ◽  
In Silico Methods

Phosphine-borane complexes are novel chemical entities with preclinical efficacy in neuronal and ophthalmic disease models. In vitro and in vivo studies showed that the metabolites of these compounds are capable of cleaving disulfide bonds implicated in the downstream effects of axonal injury. A difficulty in using standard in silico methods for studying these drugs is that most computational tools are not designed for borane-containing compounds. Using in silico and machine learning methodologies, the absorption-distribution properties of these unique compounds were assessed. Features examined with in silico methods included cellular permeability, octanol-water partition coefficient, blood-brain barrier permeability, oral absorption and serum protein binding. The resultant neural networks demonstrated an appropriate level of accuracy and were comparable to existing in silico methodologies. Specifically, they were able to reliably predict pharmacokinetic features of known boron-containing compounds. These methods predicted that phosphine-borane compounds and their metabolites meet the necessary pharmacokinetic features for orally active drug candidates. This study showed that the combination of standard in silico predictive and machine learning models with neural networks is effective in predicting pharmacokinetic features of novel boron-containing compounds as neuroprotective drugs.

scholarly journals Plant Physiology, 2021 Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7

2021 ◽  
Author(s):  
Jianghao Wu ◽  
Liwei Rong ◽  
Weijun Lin ◽  
Lingxi Kong ◽  
Dengjie Wei ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Disulfide Bonds ◽  
Kinase Activity ◽  
Light Harvesting ◽  
Photosynthetic Electron Transport ◽  
State Transitions ◽  
Light Harvesting Complex ◽  
Light Harvesting Complex Ii

Abstract In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

Silent mutations affect in vivo protein folding in Escherichia coli

2002 ◽  
Vol 293 (1) ◽  
pp. 537-541 ◽  
Author(s):  
Patricia Cortazzo ◽  
Carlos Cerveñansky ◽  
Mónica Marı́n ◽  
Claude Reiss ◽  
Ricardo Ehrlich ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Folding

scholarly journals Comprehensive assessment of post-prandial protein handling by the application of intrinsically labelled protein in vivo in human subjects

2021 ◽  
pp. 1-9
Author(s):  
Jorn Trommelen ◽  
Andrew M. Holwerda ◽  
Philippe J. M. Pinckaers ◽  
Luc J. C. van Loon
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Stable Isotope ◽  
Dietary Protein ◽  
Protein Metabolism ◽  
Muscle Protein ◽  
Human Subjects ◽  
Whole Body ◽  
Body Protein

All human tissues are in a constant state of remodelling, regulated by the balance between tissue protein synthesis and breakdown rates. It has been well-established that protein ingestion stimulates skeletal muscle and whole-body protein synthesis. Stable isotope-labelled amino acid methodologies are commonly applied to assess the various aspects of protein metabolism in vivo in human subjects. However, to achieve a more comprehensive assessment of post-prandial protein handling in vivo in human subjects, intravenous stable isotope-labelled amino acid infusions can be combined with the ingestion of intrinsically labelled protein and the collection of blood and muscle tissue samples. The combined application of ingesting intrinsically labelled protein with continuous intravenous stable isotope-labelled amino acid infusion allows the simultaneous assessment of protein digestion and amino acid absorption kinetics (e.g. release of dietary protein-derived amino acids into the circulation), whole-body protein metabolism (whole-body protein synthesis, breakdown and oxidation rates and net protein balance) and skeletal muscle metabolism (muscle protein fractional synthesis rates and dietary protein-derived amino acid incorporation into muscle protein). The purpose of this review is to provide an overview of the various aspects of post-prandial protein handling and metabolism with a focus on insights obtained from studies that have applied intrinsically labelled protein under a variety of conditions in different populations.

Folding of peptides and proteins: role of disulfide bonds, recent developments

2013 ◽  
Vol 4 (6) ◽  
pp. 597-604 ◽  
Author(s):  
Yuji Hidaka ◽  
Shigeru Shimamoto
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Difficult Problem ◽  
Chemical Methods ◽  
Mutant Proteins ◽  
Folding Intermediates ◽  
Peptide Chemistry ◽  
Recent Developments

AbstractDisulfide-containing proteins are ideal models for studies of protein folding as the folding intermediates can be observed, trapped, and separated by HPLC during the folding reaction. However, regulating or analyzing the structures of folding intermediates of peptides and proteins continues to be a difficult problem. Recently, the development of several techniques in peptide chemistry and biotechnology has resulted in the availability of some powerful tools for studying protein folding in the context of the structural analysis of native, mutant proteins, and folding intermediates. In this review, recent developments in the field of disulfide-coupled peptide and protein folding are discussed, from the viewpoint of chemical and biotechnological methods, such as analytical methods for the detection of disulfide pairings, chemical methods for disulfide bond formation between the defined Cys residues, and applications of diselenide bonds for the regulation of disulfide-coupled peptide and protein folding.

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