Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

• MeSH
• alkylace MeSH
• footprinting proteinů metody   MeSH
• halogenace MeSH
• imunokomplex chemie   MeSH
• lidé MeSH
• mapování epitopu * metody   MeSH
• monoklonální protilátky chemie   imunologie   MeSH
• receptor erbB-2 * chemie   imunologie   MeSH
• trastuzumab * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

• Klíčová slova
• Antibody, Biosimilars, Hydrogen/deuterium exchange, Mass spectrometry, Protein structure and dynamics, Proteolysis,
• MeSH
• deuterium MeSH
• hmotnostní spektrometrie MeSH
• protilátky * MeSH
• vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
• vodík MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deuterium MeSH
• protilátky * MeSH
• vodík MeSH

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Klíčová slova
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• deuterium MeSH
• hem chemie   MeSH
• hemoproteiny * MeSH
• hmotnostní spektrometrie metody   MeSH
• kyslík metabolismus   MeSH
• umělá inteligence MeSH
• vodík-deuteriová výměna metody   MeSH
• vodík chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deuterium MeSH
• hem MeSH
• hemoproteiny * MeSH
• kyslík MeSH
• vodík MeSH

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

• MeSH
• denaturace proteinů MeSH
• fosfoglycerátkinasa genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kinetika MeSH
• lidé MeSH
• sbalování proteinů * MeSH
• termodynamika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoglycerátkinasa MeSH
• PGK1 protein, human MeSH Prohlížeč

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L→G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity (≈20 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance.

• Klíčová slova
• FAD binding, allosterism, antioxidant defense, cavity-making mutation, flavoprotein, protein core, structural perturbation,
• Publikační typ
• časopisecké články MeSH

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

• Klíčová slova
• 14-3-3 protein, 70-kDa heat shock protein (Hsp70), HSP70, Hsp70, Tomm34, dimerization, hydrogen-deuterium exchange, molecular chaperone, phosphorylation, protein folding, protein import, protein kinase A (PKA), protein-nucleic acid interaction, protein–protein interaction, translocase of outer mitochondrial membrane 34 (TOMM34),
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosforylace fyziologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mitochondriální importní komplex MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• molekulární chaperony metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• proteiny tepelného šoku HSP70 metabolismus   MeSH
• proteiny tepelného šoku HSP72 metabolismus   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• signální transdukce MeSH
• transkripční faktory genetika   metabolismus   MeSH
• transportní proteiny mitochondriální membrány genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BCL2-associated athanogene 1 protein MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• mitochondriální importní komplex MeSH
• mitochondriální proteiny MeSH
• molekulární chaperony MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 MeSH
• proteiny tepelného šoku HSP70 MeSH
• proteiny tepelného šoku HSP72 MeSH
• proteiny tepelného šoku HSP90 MeSH
• TOMM34 protein, human MeSH Prohlížeč
• transkripční faktory MeSH
• transportní proteiny mitochondriální membrány MeSH

Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.

• Klíčová slova
• Src family kinases, Src phosphorylation, adhesion, anterior gradient proteins, cancer, migration, secreted protein disulfide isomerase family,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Increased activity of the chaperones Hsp70 and Hsp90 is a common feature of solid tumours. Translocase of the outer mitochondrial membrane 34 (Tomm34) is a cochaperone of both Hsp70 and Hsp90 that was found to be overexpressed in colorectal, hepatocellular, lung and breast carcinomas. The expression profile of Tomm34 in ovarian cancer has not been investigated. Therefore, the aim of the current study was to investigate the expression pattern of Tomm34 in ovarian carcinomas and analyse its correlation with clinico-pathological parameters. RESULTS: Epithelial ovarian cancers (140) were histologically classified based on their morphology and graded into two types comprising 5 histologic subgroups. Type I carcinomas comprise low grade serous (LGSC), clear cell (CCOC) and endometrioid (ENOC), type II comprises high grade serous carcinomas (HGSC) and solid, pseudoendometrioid, transitional carcinomas (SET). Tomm34 was more highly expressed in type II than type I carcinomas (p < 0.0001). Comparing tumours based on the mutation in the TP53 gene revealed similar results, where mutant tumours exhibited significantly higher levels of Tomm34 (p < 0.0001). The decreased levels of Tomm34 in type I carcinomas were particularly evident in clear cell and mucinous carcinomas. The expression of Tomm34 was also positively correlated with FIGO stage (r = 0.23; p = 0.007). Tomm34 levels also indicated poor prognosis for patients with mutant p53. CONCLUSIONS: Our data indicate that Tomm34 is commonly expressed at high levels in epithelial ovarian cancers, except for the clear cell and mucinous subtypes. The expression of Tomm34 corresponds with the dualistic model of ovarian cancer pathogenesis where high grade, type II tumours exhibit higher expression of Tomm34 in contrast to type I tumours. These data are also comparable to the previous findings that Tomm34 is a marker of progression and poor prognosis in human cancer.

• Klíčová slova
• Chaperone, Epithelial ovarian cancer, Heat shock protein, Immunohistochemistry, Ovary, Tomm34, Tumour,
• MeSH
• dospělí MeSH
• epiteliální ovariální karcinom metabolismus   patologie   MeSH
• imunohistochemie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mitochondriální importní komplex MeSH
• mutace MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nádory vaječníků metabolismus   patologie   MeSH
• prognóza MeSH
• progrese nemoci MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitochondriální importní komplex MeSH
• nádorové biomarkery MeSH
• nádorový supresorový protein p53 MeSH
• TOMM34 protein, human MeSH Prohlížeč
• TP53 protein, human MeSH Prohlížeč
• transportní proteiny mitochondriální membrány MeSH

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

• Klíčová slova
• Allostery, Chaperone, Cochaperone, Mass Spectrometry, Protein Conformation, Protein Structure, Protein-Protein Interactions, Structural Biology,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• fyziologický stres MeSH
• HEK293 buňky MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• mitochondriální importní komplex MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteiny tepelného šoku HSP70 chemie   metabolismus   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• mitochondriální importní komplex MeSH
• proteiny tepelného šoku HSP70 MeSH
• STUB1 protein, human MeSH Prohlížeč
• TOMM34 protein, human MeSH Prohlížeč
• transportní proteiny mitochondriální membrány MeSH
• ubikvitinligasy MeSH

BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.

• Klíčová slova
• LC-SWATH-MS/MS, PDLIM2, SPR, protein-protein interactions, pull-down assay,
• MeSH
• chromatografie kapalinová MeSH
• interakční proteinové domény a motivy genetika   MeSH
• lidé MeSH
• mikrofilamentové proteiny genetika   izolace a purifikace   MeSH
• nádory genetika   patologie   MeSH
• povrchová plasmonová rezonance * MeSH
• proteiny s doménou LIM genetika   izolace a purifikace   MeSH
• streptavidin chemie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikrofilamentové proteiny MeSH
• PDLIM2 protein, human MeSH Prohlížeč
• proteiny s doménou LIM MeSH
• streptavidin MeSH