TY - JOUR AU - Szabó, Eszter AU - Nemes-Nikodém, Éva AU - Vass, Krisztina Rubina AU - Zámbó, Zsófia Melinda AU - Zrupko, E. AU - Törőcsik, Beáta AU - Ozohanics, Olivér AU - Nagy, Bálint AU - Ambrus, Attila TI - Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 24 PY - 2023 IS - 13 PG - 23 SN - 1661-6596 DO - 10.3390/ijms241310826 UR - https://m2.mtmt.hu/api/publication/34075046 ID - 34075046 N1 - Export Date: 1 September 2023 Correspondence Address: Ambrus, A.; Department of Biochemistry, 37-47 Tuzolto St, Hungary; email: ambrus.attila@med.semmelweis-univ.hu Funding details: TKP2021-EGA-25, ÚNKP-19-3-III-SE-17 Funding details: Horizon 2020 Framework Programme, H2020, PID18322 Funding details: Helmholtz-Zentrum Berlin für Materialien und Energie, HZB Funding details: Semmelweis Egyetem, STIA-OTKA-2021 Funding details: Hungarian Scientific Research Fund, OTKA, 143627 Funding details: Helmholtz-Zentrum für Umweltforschung, UFZ Funding details: Nemzeti Kutatási, Fejlesztési és Innovaciós Alap, NKFIA Funding text 1: This research was funded by the Hungarian Brain Research Program 2 (2017-1.2.1-NKP-2017-00002 grant, to Vera Adam-Vizi), Semmelweis University (STIA-OTKA-2021 grant, to A.A.), Hungarian Scientific Research Fund (OTKA grant 143627, to A.A.), Ministry of Innovation and Technology of Hungary (TKP2021-EGA-25 grant, to A.A. and ÚNKP-19-3-III-SE-17 grant of the New National Excellence Program, to E.S.; project no. TKP2021-EGA-25 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA funding scheme), and European Union’s Horizon 2020 Research and Innovation Programme (Structural Biology Research Infrastructures for Translational Research and Discovery, iNEXT-Discovery PID18322 and MX-212-00269-ST (Helmholtz Zentrum Berlin/CALIPSO) grants, both to A.A.). Funding text 2: The authors gratefully acknowledge the generous support from Vera Adam-Vizi (Semmelweis University) and Manfred S. Weiss (Helmholtz-Zentrum Berlin). AB - Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the forward and reverse catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised. None of the above three variants lent themselves to an in-depth structural analysis via X-ray crystallography due to inherent protein instability. Crystal structures at 2.89 and 2.44 Å resolutions were determined for the I318T- and I358T-hLADH variants, respectively; structure analysis revealed minor conformational perturbations, which correlated well with the residual LADH activities, in both cases. For the dimer interface variants G426E-, I445M-, and R447G-hLADH, enzyme activities and FAD loss were determined and compared against the previously published structural data. © 2023 by the authors. LA - English DB - MTMT ER - TY - JOUR AU - Nagy, Bálint AU - Polak, Martin AU - Ozohanics, Olivér AU - Zámbó, Zsófia Melinda AU - Szabó, Eszter AU - Hubert, Ágnes AU - Jordan, Frank AU - Novaček, Jiří AU - Ádám, Veronika AU - Ambrus, Attila TI - Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry. Implications for the overall hKGDHc structure. TS - Implications for the overall hKGDHc structure. JF - BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS J2 - BBA-GEN SUBJECTS VL - 1865 PY - 2021 IS - 6 PG - 17 SN - 0304-4165 DO - 10.1016/j.bbagen.2021.129889 UR - https://m2.mtmt.hu/api/publication/31917407 ID - 31917407 N1 - Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary Central European Institute of Technology, Masaryk University, Brno, Czech Republic Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ, United States Export Date: 18 March 2021 CODEN: BBGSB Correspondence Address: Ambrus, A.; Department of Biochemistry, Budapest, 37-47 Tuzolto St., Hungary; email: ambrus.attila@med.semmelweis-univ.hu Funding details: National Institutes of Health, NIH, GM050380, LM2018127, R15-GM116077 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding details: Semmelweis Egyetem Funding details: Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: 61826 690289, 61830Z0100 EATV Funding details: 2017-1.2.1-NKP-2017-00002, KTIA_13_NAP-A-III/6 Funding text 1: Financial support was secured from the Hungarian Academy of Sciences (MTA grant 02001 to V.A-V.), Hungarian Scientific Research Fund (OTKA grant 112230 to V.A-V.), Hungarian Brain Research Program (grants KTIA_13_NAP-A-III/6 and 2017-1.2.1-NKP-2017-00002 to V.A-V.), Young Investigator Research Grants of Semmelweis University and Chemical Works of Gedeon Richter Plc. (to A.A.), Hungarian Higher Education Institution Excellence Program (FIKP grants 61826 690289 EATV and 61830Z0100 EATV to A.A.) and National Institutes of Health (grants GM050380 and R15-GM116077 to F. J.). CIISB research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography. Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary Central European Institute of Technology, Masaryk University, Brno, Czech Republic Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ, United States Export Date: 29 March 2021 CODEN: BBGSB Correspondence Address: Ambrus, A.; Department of Biochemistry, Budapest, 37-47 Tuzolto St., Hungary; email: ambrus.attila@med.semmelweis-univ.hu Funding details: National Institutes of Health, NIH, GM050380, LM2018127, R15-GM116077 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding details: Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: 61826 690289, 61830Z0100 EATV Funding text 1: Financial support was secured from the Hungarian Academy of Sciences (MTA grant 02001 to V.A-V.), Hungarian Scientific Research Fund (OTKA grant 112230 to V.A-V.), Hungarian Brain Research Program (grants KTIA_13_NAP-A-III/6 and 2017-1.2.1-NKP-2017-00002 to V.A-V.), Young Investigator Research Grants of Semmelweis University and Chemical Works of Gedeon Richter Plc . (to A.A.), Hungarian Higher Education Institution Excellence Program (FIKP grants 61826 690289 EATV and 61830Z0100 EATV to A.A.) and National Institutes of Health (grants GM050380 and R15-GM116077 to F. J.). CIISB research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography. Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary Central European Institute of Technology, Masaryk University, Brno, Czech Republic Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ, United States Export Date: 30 March 2021 CODEN: BBGSB Correspondence Address: Ambrus, A.; Department of Biochemistry, Budapest, 37-47 Tuzolto St., Hungary; email: ambrus.attila@med.semmelweis-univ.hu Funding details: National Institutes of Health, NIH, GM050380, LM2018127, R15-GM116077 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding details: Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: 61826 690289, 61830Z0100 EATV Funding text 1: Financial support was secured from the Hungarian Academy of Sciences (MTA grant 02001 to V.A-V.), Hungarian Scientific Research Fund (OTKA grant 112230 to V.A-V.), Hungarian Brain Research Program (grants KTIA_13_NAP-A-III/6 and 2017-1.2.1-NKP-2017-00002 to V.A-V.), Young Investigator Research Grants of Semmelweis University and Chemical Works of Gedeon Richter Plc . (to A.A.), Hungarian Higher Education Institution Excellence Program (FIKP grants 61826 690289 EATV and 61830Z0100 EATV to A.A.) and National Institutes of Health (grants GM050380 and R15-GM116077 to F. J.). CIISB research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography. AB - The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers.We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling.We report the 2.9 Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1-150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc.The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly.The revealed structural information will support the future pharmacologically targeting of the hKGDHc. LA - English DB - MTMT ER - TY - JOUR AU - Nagy, Bálint AU - Martin, Polak AU - Ozohanics, Olivér AU - Zámbó, Zsófia Melinda AU - Szabó, Eszter AU - Hubert, Ágnes AU - Frank, Jordan AU - Jiri, Novacek AU - Ádám, Veronika AU - Ambrus, Attila TI - Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure JF - PROTEIN SCIENCE J2 - PROTEIN SCI VL - 30 PY - 2021 IS - S1 SP - 171 EP - 171 PG - 1 SN - 0961-8368 DO - 10.1002/pro.4191 UR - https://m2.mtmt.hu/api/publication/33081530 ID - 33081530 N1 - Award Winners and Abstracts of the 35th Anniversary Symposium of The Protein Society, July 7–14, 2021 | Virtual LA - English DB - MTMT ER - TY - JOUR AU - Nagy, Bálint AU - Polak, Martin AU - Ozohanics, Olivér AU - Zámbó, Zsófia Melinda AU - Szabó, Eszter AU - Hubert, Ágnes AU - Jordan, Frank AU - Novacek, Jiri AU - Ádám, Veronika AU - Ambrus, Attila TI - Structure of the Dihydrolipoamide Succinyltransferase (E2) Component of the Human α-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and Cross-linking mass spectrometry: Implications for the overall hKGDHc structure JF - FREE RADICAL BIOLOGY AND MEDICINE J2 - FREE RADICAL BIO MED VL - 159 PY - 2020 IS - Suppl. 1 SP - S29 EP - S30 SN - 0891-5849 DO - 10.1016/j.freeradbiomed.2020.10.088 UR - https://m2.mtmt.hu/api/publication/33081442 ID - 33081442 LA - English DB - MTMT ER - TY - JOUR AU - Nagy, Bálint AU - Ambrus, Attila AU - Zámbó, Zsófia Melinda AU - Hubert, Ágnes AU - Polak, Martin AU - Szabó, Eszter AU - Novacek, Jiri AU - Jordan, Frank AU - Ádám, Veronika TI - CRYO-EM STRUCTURE OF THE DIHYDROLIPOAMIDE SUCCINYLTRANSFERASE (E2) COMPONENT OF THE HUMAN ALPHA-KETOGLUTARATE DEHYDROGENASE COMPLEX JF - PROTEIN SCIENCE J2 - PROTEIN SCI VL - 28 PY - 2019 SP - 155 EP - 155 PG - 1 SN - 0961-8368 UR - https://m2.mtmt.hu/api/publication/31138483 ID - 31138483 N1 - Supplement: 1 LA - English DB - MTMT ER - TY - JOUR AU - Szabó, Eszter AU - Wilk, Piotr AU - Nagy, Bálint AU - Mizsei, Reka AU - Zámbó, Zsófia Melinda AU - Bui, David AU - Weichsel, Andrzej AU - Arjunan, Palaniappa AU - Torocsik, Beata AU - Hubert, Ágnes AU - Furey, William AU - Monfort, William AU - Jordan, Frank AU - Weiss, Manfred AU - Ádám, Veronika AU - Ambrus, Attila TI - MOLECULAR MECHANISMS GIVING RISE TO HUMAN DIHYDROLIPOAMIDE DEHYDROGENASE DEFICIENCY - STRUCTURAL ANALYSIS OF SEVEN DISEASE-RELEVANT ENZYME VARIANTS JF - PROTEIN SCIENCE J2 - PROTEIN SCI VL - 28 PY - 2019 SP - 163 EP - 164 PG - 2 SN - 0961-8368 UR - https://m2.mtmt.hu/api/publication/31138484 ID - 31138484 N1 - Supplement: 1 LA - English DB - MTMT ER - TY - JOUR AU - Szabó, Eszter AU - Wilk, Piotr AU - Nagy, Bálint AU - Zámbó, Zsófia Melinda AU - Bui, Dávid AU - Weichsel, Andrzej AU - Arjunan, Palaniappa AU - Törőcsik, Beáta AU - Hubert, Agnes AU - Furey, William AU - Montfort, William R AU - Jordan, Frank AU - Weiss, Manfred S AU - Ádám, Veronika AU - Ambrus, Attila TI - Underlying molecular alterations in human dihydrolipoamide dehydrogenase deficiency revealed by structural analyses of disease-causing enzyme variants JF - HUMAN MOLECULAR GENETICS J2 - HUM MOL GENET VL - 28 PY - 2019 IS - 20 SP - 3339 EP - 3354 PG - 16 SN - 0964-6906 DO - 10.1093/hmg/ddz177 UR - https://m2.mtmt.hu/api/publication/30799456 ID - 30799456 AB - Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G, and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes, and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches. LA - English DB - MTMT ER - TY - JOUR AU - Szabó, Eszter AU - Mizsei, Réka AU - Wilk, P AU - Zámbó, Zsófia Melinda AU - Törőcsik, Beáta AU - Weiss, MS AU - Ádám, Veronika AU - Ambrus, Attila TI - Crystal structures of the disease-causing D444V mutant and the relevant wild type human dihydrolipoamide dehydrogenase JF - FREE RADICAL BIOLOGY AND MEDICINE J2 - FREE RADICAL BIO MED VL - 124 PY - 2018 SP - 214 EP - 220 PG - 7 SN - 0891-5849 DO - 10.1016/j.freeradbiomed.2018.06.008 UR - https://m2.mtmt.hu/api/publication/3407990 ID - 3407990 N1 - Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, H-1094, Hungary Macromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, D-12489, Berlin, Germany Cited By :2 Export Date: 24 January 2020 CODEN: FRBME Correspondence Address: Ambrus, A.; Department of Medical Biochemistry, Semmelweis University, 37-47 Tuzolto Street, Hungary; email: ambrus.attila@med.semmelweis-univ.hu Chemicals/CAS: dihydrolipoamide dehydrogenase, 37340-89-9, 9001-18-7; Dihydrolipoamide Dehydrogenase; Mutant Proteins Funding details: Hungarian Scientific Research Fund Funding details: Hungarian Scientific Research Fund, 112230 Funding details: Magyar Tudományos Akadémia, 02001 Funding text 1: Financial support was secured from the Hungarian Academy of Sciences (MTA grant 02001 to V.A-V.), the Hungarian Scientific Research Fund (OTKA grant 112230 to V.A-V.), the Hungarian Brain Research Program (grants KTIA_13_NAP-A-III/6. and 2017-1.2.1-NKP-2017-00002 to V.A-V.), the EMBO , Fulbright , and Bolyai Fellowships (to A.A.), and the Young Investigator Research Grants of the Semmelweis University and Gedeon Richter Pharmaceuticals PIc . (to A.A.). Data collection at Helmholtz-Zentrum Berlin was carried out under contract number 16204087-ST. Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, H-1094, Hungary Macromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, D-12489, Berlin, Germany Cited By :3 Export Date: 4 January 2021 CODEN: FRBME Correspondence Address: Ambrus, A.; Department of Medical Biochemistry, Semmelweis University, 37-47 Tuzolto Street, Hungary; email: ambrus.attila@med.semmelweis-univ.hu Chemicals/CAS: dihydrolipoamide dehydrogenase, 37340-89-9, 9001-18-7; Dihydrolipoamide Dehydrogenase; Mutant Proteins Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding text 1: Financial support was secured from the Hungarian Academy of Sciences (MTA grant 02001 to V.A-V.), the Hungarian Scientific Research Fund (OTKA grant 112230 to V.A-V.), the Hungarian Brain Research Program (grants KTIA_13_NAP-A-III/6. and 2017-1.2.1-NKP-2017-00002 to V.A-V.), the EMBO , Fulbright , and Bolyai Fellowships (to A.A.), and the Young Investigator Research Grants of the Semmelweis University and Gedeon Richter Pharmaceuticals PIc . (to A.A.). Data collection at Helmholtz-Zentrum Berlin was carried out under contract number 16204087-ST. Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, H-1094, Hungary Macromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, D-12489, Berlin, Germany Cited By :3 Export Date: 5 January 2021 CODEN: FRBME Correspondence Address: Ambrus, A.; Department of Medical Biochemistry, Semmelweis University, 37-47 Tuzolto Street, Hungary; email: ambrus.attila@med.semmelweis-univ.hu Chemicals/CAS: dihydrolipoamide dehydrogenase, 37340-89-9, 9001-18-7; Dihydrolipoamide Dehydrogenase; Mutant Proteins Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding text 1: Financial support was secured from the Hungarian Academy of Sciences (MTA grant 02001 to V.A-V.), the Hungarian Scientific Research Fund (OTKA grant 112230 to V.A-V.), the Hungarian Brain Research Program (grants KTIA_13_NAP-A-III/6. and 2017-1.2.1-NKP-2017-00002 to V.A-V.), the EMBO , Fulbright , and Bolyai Fellowships (to A.A.), and the Young Investigator Research Grants of the Semmelweis University and Gedeon Richter Pharmaceuticals PIc . (to A.A.). Data collection at Helmholtz-Zentrum Berlin was carried out under contract number 16204087-ST. AB - We report the crystal structures of the human (dihydro)lipoamide dehydrogenase (hLADH, hE3) and its disease-causing homodimer interface mutant D444V-hE3 at 2.27 and 1.84 Å resolution, respectively. The wild type structure is a unique uncomplexed, unliganded hE3 structure with the true canonical sequence. Based on the structural information a novel molecular pathomechanism is proposed for the impaired catalytic activity and enhanced capacity for reactive oxygen species generation of the pathogenic mutant. The mechanistic model involves a previously much ignored solvent accessible channel leading to the active site that might be perturbed also by other disease-causing homodimer interface substitutions of this enzyme. © 2018 Elsevier Inc. LA - English DB - MTMT ER - TY - JOUR AU - Ambrus, Attila AU - Wang, J AU - Mizsei, Réka AU - Zámbó, Zsófia Melinda AU - Törőcsik, Beáta AU - Jordan, F AU - Ádám, Veronika TI - Structural alterations induced by ten disease-causing mutations of human dihydrolipoamide dehydrogenase analyzed by hydrogen/deuterium-exchange mass spectrometry: Implications for the structural basis of E3 deficiency JF - BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE J2 - BBA-MOL BASIS DIS VL - 1862 PY - 2016 IS - 11 SP - 2098 EP - 2109 PG - 12 SN - 0925-4439 DO - 10.1016/j.bbadis.2016.08.013 UR - https://m2.mtmt.hu/api/publication/3114546 ID - 3114546 N1 - Attila Ambrus, Junjie Wang and Reka Mizsei contributed equally to this work. Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, Hungary Department of Chemistry, Rutgers University, Newark, NJ, United States Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, United States Laboratory of Immunobiology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, United States Cited By :9 Export Date: 28 May 2020 CODEN: BBADE Correspondence Address: Ambrus, A.; Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis UniversityHungary; email: ambrus.attila@med.semmelweis-univ.hu Chemicals/CAS: amino acid, 65072-01-7; deuterium, 7782-39-0; dihydrolipoamide dehydrogenase, 37340-89-9, 9001-18-7; hydrogen, 12385-13-6, 1333-74-0 Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding text 1: We are grateful to Drs. Oliver Ozohanics, Karoly Vekey (both from the Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary) and Arpad Somogyi (Ohio State University, Columbus, OH, USA) for their contributions to the method development in mass spectrometry. Financial support is gratefully acknowledged from the Hungarian Academy of Sciences (MTA grant 02001 to V.A.-V.), the Hungarian Scientific Research Fund (OTKA, grant 112230 to V.A.-V.), the Hungarian Brain Research Program (grant KTIA_13_NAP-A-III/6 to V.A.-V.), the Bolyai and the Fulbright Fellowships (to A.A.), NIH - R15GM116077 and NSF-CHE - 1402675 (to F.J.). Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, Hungary Department of Chemistry, Rutgers University, Newark, NJ, United States Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, United States Laboratory of Immunobiology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, United States Cited By :12 Export Date: 5 January 2021 CODEN: BBADE Correspondence Address: Ambrus, A.; Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis UniversityHungary; email: ambrus.attila@med.semmelweis-univ.hu Chemicals/CAS: amino acid, 65072-01-7; deuterium, 7782-39-0; dihydrolipoamide dehydrogenase, 37340-89-9, 9001-18-7; hydrogen, 12385-13-6, 1333-74-0 Funding details: Hungarian Scientific Research Fund, OTKA Funding details: Magyar Tudományos Akadémia, MTA Funding details: Hungarian Scientific Research Fund, OTKA, 112230 Funding details: Magyar Tudományos Akadémia, MTA, 02001 Funding text 1: We are grateful to Drs. Oliver Ozohanics, Karoly Vekey (both from the Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary) and Arpad Somogyi (Ohio State University, Columbus, OH, USA) for their contributions to the method development in mass spectrometry. Financial support is gratefully acknowledged from the Hungarian Academy of Sciences (MTA grant 02001 to V.A.-V.), the Hungarian Scientific Research Fund (OTKA, grant 112230 to V.A.-V.), the Hungarian Brain Research Program (grant KTIA_13_NAP-A-III/6 to V.A.-V.), the Bolyai and the Fulbright Fellowships (to A.A.), NIH - R15GM116077 and NSF-CHE - 1402675 (to F.J.). AB - Pathogenic amino acid substitutions of the common E3 component (hE3) of the human alpha-ketoglutarate dehydrogenase and the pyruvate dehydrogenase complexes lead to severe metabolic diseases (E3 deficiency), which usually manifest themselves in cardiological and/or neurological symptoms and often cause premature death. To date, 14 disease-causing amino acid substitutions of the hE3 component have been reported in the clinical literature. None of the pathogenic protein variants has lent itself to high-resolution structure elucidation by X-ray or NMR. Hence, the structural alterations of the hE3 protein caused by the disease-causing mutations and leading to dysfunction, including the enhanced generation of reactive oxygen species by selected disease-causing variants, could only be speculated. Here we report results of an examination of the effects on the protein structure of ten pathogenic mutations of hE3 using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), a new and state-of-the-art approach of solution structure elucidation. On the basis of the results, putative structural and mechanistic conclusions were drawn regarding the molecular pathogenesis of each disease-causing hE3 mutation addressed in this study. © 2016 Elsevier B.V. LA - English DB - MTMT ER - TY - JOUR AU - Nagy, Bálint AU - Hubert, Ágnes AU - Szabó, Eszter AU - Zámbó, Zsófia Melinda AU - Ádám, Veronika AU - Ambrus, Attila TI - Isolation and crystallization of the pathological mutants of human dihydrolipoamide dehydrogenase JF - BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS J2 - BBA-BIOENERGETICS VL - 1857 PY - 2016 IS - Suppl. SP - e42 SN - 0005-2728 DO - 10.1016/j.bbabio.2016.04.311 UR - https://m2.mtmt.hu/api/publication/3262089 ID - 3262089 LA - English DB - MTMT ER - TY - JOUR AU - Szabó, Eszter AU - Mizsei, R AU - Zámbó, Zsófia Melinda AU - Torocsik, B AU - Weiss, MS AU - Adam-Vizi, V AU - Ambrus, Attila TI - Crystal structure of the D444V disease-causing mutant of human dihydrolipoamide dehydrogenase JF - PROTEIN SCIENCE J2 - PROTEIN SCI VL - 25 PY - 2016 IS - S1 SP - 162 EP - 162 PG - 1 SN - 0961-8368 UR - https://m2.mtmt.hu/api/publication/3262113 ID - 3262113 LA - English DB - MTMT ER - TY - JOUR AU - Szabó, Eszter AU - Mizsei, Réka AU - Zámbó, Zsófia Melinda AU - Törőcsik, Beáta AU - Weiss, Manfred S AU - Ádám, Veronika AU - Ambrus, Attila TI - Crystal structure of the D444V disease-causing mutant of human dihydrolipoamide dehydrogenase JF - BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS J2 - BBA-BIOENERGETICS VL - 1857 PY - 2016 IS - Suppl. SP - e100 SN - 0005-2728 DO - 10.1016/j.bbabio.2016.04.337 UR - https://m2.mtmt.hu/api/publication/3262509 ID - 3262509 LA - English DB - MTMT ER - TY - JOUR AU - Lajkó, Eszter AU - Bányai, Péter AU - Zámbó, Zsófia Melinda AU - Kursinszki, László AU - Szőke, Éva AU - Kőhidai, László TI - Targeted tumor therapy by Rubia tinctorum L.: analytical characterization of hydroxyanthraquinones and investigation of their selective cytotoxic, adhesion and migration modulator effects on melanoma cell lines (A2058 and HT168‑M1) JF - CANCER CELL INTERNATIONAL J2 - CANCER CELL INT VL - 15 PY - 2015 PG - 15 SN - 1475-2867 DO - 10.1186/s12935-015-0271-4 UR - https://m2.mtmt.hu/api/publication/3003992 ID - 3003992 AB - Background: Alizarin and purpurin are di- and trihydroxyanthraquinones derived from Rubia tinctorum L. Previous pharmacological studies have demonstrated that they exhibit certain degree of selective inhibitory effects towards cancer cells suggesting their application as a targeted drug for cancer. Our present work was aimed to investigate the suitability of hydroxyanthraquinones of Rubia tinctorum L. for targeted tumor therapy. The effects of alizarin, purpurin and an aqueous extract from transformed hairy root culture of Rubia tinctorum L. were examined on (1) cell proliferation, (2) apoptosis, (3) cell adhesion/morphology and (4) migration (chemotaxis, chemokinesis) of human melanoma cell lines (A2058, HT168-M1) and human fibroblast cells (MRC-5), as well as (5) the aqueous extract was analytically characterized. Methods: The aqueous extract was prepared from R. tinctorum hairy root culture and qualitatively analyzed by HPLC and ESI-MS methods. The cell growth inhibitory activity of anthraquinones was evaluated by MTT-assay and by flow cytometry. The effect of anthraquinones on cell adhesion was measured by an impedance based technique, the xCELLigence SP. For the chemotaxis assay NeuroProbe® chamber was used. Computer based holographic microscopy was applied to analyze chemokinetic responses as well as morphometry. Statistical significance was determined by the one-way ANOVA test. Results: In the aqueous extract, munjistin (Mr = 284, tR = 18.4 min) as a principal component and three minor anthraquinones (pseudopurpurin, rubiadin and nordamnacanthal) were identified. The purpurin elicited a stronger but not apoptosis-mediated antitumor effect in melanoma cells (A2058: 10-6-10-5 M: 90.6-64.1 %) than in normal fibroblasts (10-6-10-5 M: 97.6-84.8 %). The aqueous extract in equimolar concentrations showed the most potent cytotoxicity after 72 h incubation (A2058: 10-6-10-5 M: 87.4-55.0 %). All tested substances elicited chemorepellent effect in melanoma cells, while in MRC-5 fibroblasts, only the alizarin exhibited such a repellent character. Indices of chemokinesis measured by holographic microscopy (migration, migration directness, motility and motility speed) were significantly enhanced by alizarin and purpurin as well, while morphometric changes were weak in the two melanoma cell lines. Conclusions: Our results highlight the effective and selective inhibitory activity of purpurin towards melanoma cells and its possible use as a targeted anticancer agent. The anthraquinones of the cytotoxic extract are suggested to apply in drug delivery systems as an anticancer drug. © 2015 Lajkó et al. LA - English DB - MTMT ER -