Effect of methylglyoxal modification on stress-induced aggregation of client proteins and their chaperoning by human αA-crystallin

α-Crystallin prevents protein aggregation under various stress conditions through its chaperone-like properties. Previously, we demonstrated that MGO (methylglyoxal) modification of αA-crystallin enhances its chaperone function and thus may affect transparency of the lens. During aging of the lens, not only αA-crystallin, but its client proteins are also likely to be modified by MGO. We have investigated the role of MGO modification of four model client proteins (insulin, α-lactalbumin, alcohol dehydrogenase and γ-crystallin) in their aggregation and structure and the ability of human αA-crystallin to chaperone them. We found that MGO modification (10–1000 μM) decreased the chemical aggregation of insulin and α-lactalbumin and thermal aggregation of alcohol dehydrogenase and γ-crystallin. Surface hydrophobicity in MGO-modified proteins decreased slightly relative to unmodified proteins. HPLC and MS analyses revealed argpyrimidine and hydroimidazolone in MGO-modified client proteins. The degree of chaperoning by αA-crystallin towards MGO-modified and unmodified client proteins was similar. Co-modification of client proteins and αA-crystallin by MGO completely inhibited stress-induced aggregation of client proteins. Our results indicate that minor modifications of client proteins and αA-crystallin by MGO might prevent protein aggregation and thus help maintain transparency of the aging lens.

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Prevention of protein aggregation by extracellular fungal sucrase of Termitomyces clypeatus

Turkish Journal of Biochemistry ◽

10.1515/tjb-2016-0297 ◽

2017 ◽

Vol 42 (3) ◽

Author(s):

Sudeshna Chowdhury ◽

Samudra Prosad Banik ◽

Rajib Majumder ◽

Shakuntala Ghorai ◽

Swagata Pal ◽

...

Keyword(s):

Protein Aggregation ◽

Protein Misfolding ◽

Structural Changes ◽

Whey Proteins ◽

Gel Filtration ◽

Molecular Size ◽

Thermal Aggregation ◽

Induced Aggregation ◽

Important Enzyme

AbstractObjective:Extracellular sucrase fromMethods:MALDI-TOF and dynamic light scattering were used to assess the monomeric and aggregated molecular size of sucrase. Thermostatted spectrophotometric assays, gel filtration assays were used to study protein aggregation. Fluorescence of bound ANS was used to monitor temperature induced structural changes in sucrase together with determination of melting temperature.Results:The mass of the monomeric unit of sucrase as 6649 Da. Enzyme inhibited DTT induced aggregation of insulin and suppressed the thermal aggregation of carbonic anhydrase, ADH and whey proteins, respectively by 83%, 68% and 70% at 70°C. Sucrase also protected about 84% activity of ADH.Conclusion:An extracellular fungal sucrase with a low monomeric size can efficiently prevent protein aggregation. The studies can impart knowledge about potential therapeutic applications of this industrially important enzyme in protein misfolding disorders.

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Super Spy variants implicate flexibility in chaperone action

eLife ◽

10.7554/elife.01584 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 34

Author(s):

Shu Quan ◽

Lili Wang ◽

Evgeniy V Petrotchenko ◽

Karl AT Makepeace ◽

Scott Horowitz ◽

...

Keyword(s):

Protein Function ◽

Genetic Selection ◽

Chaperone Activity ◽

Wild Type ◽

Good Relationship ◽

Chaperone Function ◽

Binding Partners ◽

Client Proteins ◽

Adaptive Recognition

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function.

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On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

10.1055/s-0038-1652598 ◽

1981 ◽

Author(s):

M Yamamoto ◽

K Watanabe ◽

Y Ando ◽

H Iri ◽

N Fujiyama ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Coagulation Factors ◽

Marked Enhancement ◽

Matrix Bound ◽

Induced Aggregation ◽

Aggregation Of Platelets ◽

Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.

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Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653405 ◽

1981 ◽

Vol 46 (02) ◽

pp. 538-542 ◽

Cited By ~ 42

Author(s):

R Pilo ◽

D Aharony ◽

A Raz

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Unsaturated Fatty Acids ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Arachidonic Acid Metabolism ◽

Ionophore A23187 ◽

Low Concentrations ◽

Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

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Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657015 ◽

1979 ◽

Vol 42 (04) ◽

pp. 1193-1206 ◽

Cited By ~ 9

Author(s):

Barbara Nunn

Keyword(s):

Platelet Aggregation ◽

Time Course ◽

Essential Role ◽

Atp Release ◽

Adenosine Diphosphate ◽

Human Platelets ◽

High Doses ◽

Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

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Faculty Opinions recommendation of Novel antioxidant role of alcohol dehydrogenase E from Escherichia coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1015362.196512 ◽

2003 ◽

Author(s):

Joan Broderick

Keyword(s):

Escherichia Coli ◽

Alcohol Dehydrogenase

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Thiol-based switching mechanisms of stress-sensing chaperones

Biological Chemistry ◽

10.1515/hsz-2020-0262 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Kathrin Ulrich ◽

Blanche Schwappach ◽

Ursula Jakob

Keyword(s):

Oxidative Stress ◽

Environmental Changes ◽

Stress Conditions ◽

Stress Exposure ◽

Atp Levels ◽

Protein Functions ◽

Activation Mechanisms ◽

Client Proteins ◽

Stress Sensing ◽

Redox Switches

AbstractThiol-based redox switches evolved as efficient post-translational regulatory mechanisms that enable individual proteins to rapidly respond to sudden environmental changes. While some protein functions need to be switched off to save resources and avoid potentially error-prone processes, protective functions become essential and need to be switched on. In this review, we focus on thiol-based activation mechanisms of stress-sensing chaperones. Upon stress exposure, these chaperones convert into high affinity binding platforms for unfolding proteins and protect cells against the accumulation of potentially toxic protein aggregates. Their chaperone activity is independent of ATP, a feature that becomes especially important under oxidative stress conditions, where cellular ATP levels drop and canonical ATP-dependent chaperones no longer operate. Vice versa, reductive inactivation and substrate release require the restoration of ATP levels, which ensures refolding of client proteins by ATP-dependent foldases. We will give an overview over the different strategies that cells evolved to rapidly increase the pool of ATP-independent chaperones upon oxidative stress and provide mechanistic insights into how stress conditions are used to convert abundant cellular proteins into ATP-independent holding chaperones.

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