Összes szerző

Kellermayer Miklós S.Z.

az alábbi absztraktok szerzői között szerepel:

Csányi Mária Csilla
Nanosurgical manipulation of extended von Willebrand factor multimers

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P07

Nanosurgical manipulation of extended von Willebrand factor multimers

Mária Csilla Csányi¹, Dominik Sziklai¹, Tímea Feller², Jolán Hársfalvi¹ and Miklós S.Z. Kellermayer¹

¹ Semmelweis University, Faculty of Medicine, Department of Biophysics and Radiation Biology

² University of Leeds, Leeds Institute of Cardiovascular and Metabolic Medicine, Discovery and Translational Science Department

The von Willebrand factor (VWF) is a varying-length multimeric chain of glycoprotein dimers/protomers with distinct domain structures. Conformational response of domains upon exposure to shear- and elongational forces ensures that VWF is able to mediate platelet adhesion to and aggregation on an injured vessel wall where it is explored or immobilized from the circulation. Human plasma-derived VWF multimers stretched by molecular combing extend through a specific hierarchy of structural intermediates of protomers as we have recently shown by using atomic force microscopy (AFM) [1]. However, the full scope of local domain stabilities and extensibilities remained hidden.

To uncover these cryptic structural details, in the present work, we used an in situ nanosurgical approach to probe whether targeted domains of VWF could be further extended and unfolded. To achieve this, surface-stabilized and pre-extended VWF multimers were manipulated at distinct spatial locations with the tip of the AFM cantilever. By moving the AFM tip in a direction perpendicular to the longitudinal axis of the VWF multimer, protein loops could be pulled out of the chain, the local extension of which was assessed following the acquisition of a subsequent topographic AFM image.

The extension resulted in ruptured and non-ruptured protein loops, with and without the appearance of hairpin-like thin sections and nodules, while the adjacent domains were non-displaced. Ruptures occurred in 27% of the VWF. Extension, which is the ratio of the length of the section following and prior to manipulation was 2.7 vs. 1.6, p= 0.0001 in non-ruptured and ruptured multimers. Nanomanipulation of protomers in which all the 5 nodules are separated, resulted in a mean final length of 345±69 nm, compared to their pre-manipulation length of 168±54 nm. None of the above results correlated with the protomer’s structural hierarchy showing the main role of the adhesion of certain domains to the mica surface. The nanosurgical manipulation used here demonstrates the VWF mechanical properties at the single domain level via extending further the C1-C6 and A1-A2-A3 domains or eventually rupturing them. The observed conformational changes indicate that VWF may have a large conformational force response potential in order to fine-tune the opening up of hidden epitopes for the different functions of the VWF.

Acknowledgment

TKP2021-EGA-23

References

[1] Csányi MC, Salamon P, Feller T, Bozó T, Hársfalvi J, Kellermayer MSZ. (2023) Protein Sci 32(1):e4535.

Hársfalvi Jolán
Biophysical Characterization of Clot Retraction in Platelet Rich Plasma of Patients with Primary Anti-phospholipid Syndrome

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P11

Biophysical Characterization of Clot Retraction in Platelet Rich Plasma of Patients with Primary Anti-phospholipid Syndrome

Jolán Hársfalvi¹_, Tímea Feller¹, György Domjan³, Klára Gadó³, Katalin Várnai², Eszter Barabás², Miklós Kellermayer¹

¹Department of Biophysical and Radiation Biology, ²Department of Laboratory Medicine and ³Department of Internal Medicine and Oncology, Semmelweis University, Budapest, Hungary

Anti-phospholipid syndrome (APS) is an autoimmune process leading to thrombotic disorders [1], but with poorly understood mechanisms.

By using atomic force microscopy (AFM)-based nano-thrombelastography (nTEG) [2], we investigated the biophysical characteristics of the fibrin network during clot formation and degradation in platelet-rich plasma (PRP) of 38 APS patients compared with 18 controls. Patients with APS and venous thromboembolism (VTE) were selected as case and control, respectively.

Citrated blood was centrifuged at room temperature (150 g, 10 min) to obtain PRP, so that platelet count was set to 50G/L. An AFM cantilever was submerged in a 0.3-mL sample and cyclically moved up and down with an amplitude of 1 μm and a speed of 1 μm/s. In addition to PRP the sample was completed with 10mM Ca²⁺, and clotting was initiated with thrombin at a final activity of 1 IU/ml. As the sample clotted, the cantilever deflected progressively during its vertical travel, reflecting the onset and increase in the elastic and viscous properties of the clot. The onset of clot formation was determined by measuring the time delay until the first non-zero force signal appeared. Clot contractility was assessed by measuring the rate of force increase. Finally, the viscoelastic response of the clot was obtained by measuring the force hysteresis area and the peak force difference in the datasets collected in the opposite cantilever directions (up versus down). The median parameter values of the APS and control samples were compared.

We found that in the APS group, the delay until the first force signal increased 2-3-fold [sec]; the slope of the force generation decreased to about 1/2 [nN/sec]; and the maximal force difference in the mechanical cycles decreased to about 1/3 [nN]. These results compare well with recent observations in which macroscale methods were used [3].

In sum, we were able to characterize quantitatively the nanoscale changes in the viscoelastic properties during platelet contractility and fibrin network formation in human PRP in a distinct pathology. We expect that the rich dataset provided by the AFM-based measurement employed here will provide insights into the molecular mechanisms associated with the pathology of APS.

References

1. Vreede AP, Bockenstedt PL, McCune WJ, Knight JS (2019) Curr Opin Rheumatol. 31(3):231-240.

2. Feller T, Kellermayer MS, Kiss B. (2014) Journal of Structural Biology 462-71

3. Le Minh, G., A.D. Peshkova, I.A. Andrianova, T.B. Sibgatullin, A.N. Maksudova, J.W. Weisel, and R. Litvinov, (2018) Clinical Science 132: 243-254

Kellermayer MIklós
Truncated titin is integrated into the human dilated cardiomyopathic sarcomere

Aug 29 - kedd

08:30 – 08:50

Molekuláris biofizika

E01

Truncated titin is integrated into the human dilated cardiomyopathic sarcomere

Dalma Kellermayer^1,2,3,$, Hedvig Tordai^2,$, Balázs Kiss², György Török², Dániel M. Péter², Alex Ali Sayour¹, Miklós Pólos¹, István Hartyánszky¹, Bálint Szilveszter¹, Siegfried Labeit⁴, Ambrus Gángó³, Gábor Bedics³, Csaba Bödör³, Tamás Radovits¹, Béla Merkely¹ and Miklós S.Z. Kellermayer²

¹Heart and Vascular Center, Semmelweis University, Budapest, Hungary

²Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

³1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

⁴DZHK Partnersite Mannheim-Heidelberg, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

Heteroyzgous truncating mutations in the TTN gene (TTNtv) encoding the giant protein titin are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here we investigated 118 clinically identified DCM human cardiac samples with next-generation sequencing, high-resolution gel electrophoresis, Western blot analysis and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene-sequence predictions were detected in the majority of the TTNtv samples. The total amount of expressed titin, which includes the truncated fragments, was comparable in the TTNtv+ and TTNtv- samples, indicating that titin haploinsufficiency may not be the leading cause of the molecular pathogenesis. Proteomic analysis of washed cardiac myofibrils and STED super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin is structurally integrated in the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape and intensity analysis pointed at structural disarrangements in the I/A junction and the M-band of TTNtv+ sarcomeres, which may play a role, via faulty mechanosensor function, in the development of manifest DCM.

Acknowledgments

This research was funded by the ÚNKP-19-3-I New National Excellence Program of The Ministry for Innovation and Technology to D.K. and grants from the Hungarian National Research, Development and Innovation Office (K135360 to M.K., FK135462 to B.K., K135076 to B.M., Project no. NVKP_16-1–2016-0017 ’National Heart Program’, and the 2020-1.1.6-JÖVŐ-2021-00013 grant) and the Thematic Excellence Programme (2020-4.1.1.-TKP2020) of the Ministry for Innovation and Technology in Hungary, within the framework of the Therapeutic Development and Bioimaging thematic programs of Semmelweis University.

Sulea Cristina
Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P27

Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Cristina M. Șulea^1,2,3, Zsolt Mártonfalvi¹, Csilla Csányi¹, Dóra Haluszka¹, Miklós Pólos^2,3, Kálmán Benke^2,3, Zoltán Szabolcs^2,3 and Miklós S. Z. Kellermayer¹

¹ Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary

² Heart and Vascular Center, Semmelweis University, 1122 Budapest, Hungary

³ Hungarian Marfan Foundation, 1122 Budapest, Hungary

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation in microfibrillar function and, conceivably, alterations in the microfibrils’ supramolecular structure.

The aim of the study was to perform a nanoscale structural characterization of fibrillin-1 microfibrils isolated from human aortic samples with different FBN1 gene mutations and to compare them with microfibrillar assemblies purified from non-MFS human aortic tissue.

Aortic wall samples were obtained from patients undergoing specific cardiovascular surgical interventions. Fibrillin-rich microfibrils were extracted by bacterial collagenase digestion and purified by size-exclusion chromatography. Atomic force microscopy was employed to visualize and study the microfibrillar assemblies.

Fibrillin-1 microfibrils displayed a characteristic “beads-on-a-string” appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50–52 nm among samples.

In conclusion, the data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-specific aortic symptomatology.

Acknowledgment

Funding sources: NRDI Office (ÚNKP-22-3-I-SE-49 to C.M.Ș.; K135360 to M.S.Z.K.; TKP2021-EGA-23), European Union (RRF-2.3.1-21-2022-00003 – National Cardiovascular Laboratory).

Török György
Adaptive changes in the a-band region of the giant protein titin in diseased human cardiac sarcomere

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P32

Adaptive changes in the A-band region of the giant protein titin in diseased human cardiac sarcomere

György Török¹, Iliza Ramazanova¹, Péter Dániel¹, András Jámbor¹, Dalma Kellermayer^1,2, Cristina M. Șulea^1,2, Zoltán Szabolcs², Miklós S. Z. Kellermayer¹ and Balázs Kiss¹

¹Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

²Heart and Vascular Center, Semmelweis University, Budapest, Hungary

Titin, the largest protein known, spans the half-sarcomere, the contractile unit of skeletal and cardiac muscle through its Z-disk to M-line and interacts with thin and thick filaments in the I- and A-band of the muscle sarcomere, respectively. Titin’s A-band segment is not well understood but is shown to be orders of magnitude less extensible than the I-band region of the molecule. Heterozygous truncating mutations (TTNtv) affecting A-band titin are often associated with dilated cardiomyopathy (DCM). Marfan syndrome (MFS), a connective tissue disorder caused by mutations of the matrix glycoprotein fibrillin is also associated with impaired cardiac contractility but its exact pathomechanism is largely unknown.

Here, we performed STED super-resolution microscopy on sections of stretched and fixed demembranated human cardiac myofibrils carrying heterozygous TTNtv mutations or on samples originated from MFS patients. Sequence-specific anti-titin antibodies included the 1) MIR, which labels titin at the ends of the thick filaments, 2) A170, labeling titin close to the M-line (this epitope is missing in TTNtv+). Sarcomere length-dependent anti-titin epitope position, shape and intensity analysis pointed at structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres. Our experiments indicate that truncated titin is able to integrate into the cardiac sarcomere. We propose that the truncated titin cannot precisely register the ends of the thick filaments, and this can ultimately lead to the manifestation of DCM by disrupting the overlapping of thin and thick filaments. In MFS sarcomeres a pronounced, ~30 nm shift away from the M-line was found in the case of the A170 titin epitope suggesting that alterations in the M-band ultrastructure might be important contributors of the impaired cardiac contractility of MFS patients.