Biochimica et biophysica acta. Proteins and proteomics最新文献

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/⁻OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/⁻OCl, 10 μM HOCl/⁻OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BβMet367, γMet264, and γMet94, identified in 10 μM HOCl/–OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups.

This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis.

Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD.

The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes widely studied for their involvement in microbial and fungal biomass degradation. The catalytic versatility of these enzymes is demonstrated by the recent discovery of LPMOs in arthropods, viruses, insects and ferns, where they fulfill diverse functions beyond biomass conversion. This mini-review puts a spotlight on a recently recognized aspect of LPMOs: their role in infectious processes in human pathogens. It discusses the occurrence and potential biological mechanisms of LPMOs associated with human pathogens and provides an outlook on future avenues in this emerging and exciting research field.

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

Prolidase (EC 3.4.13.9) is an enzyme that specifically hydrolyzes Xaa-Pro dipeptides into free amino acids. We previously studied kinetic behaviours and solved the crystal structure of wild-type (WT) Lactococcus lactis prolidase (Llprol), showing that this homodimeric enzyme has unique characteristics: allosteric behaviour and substrate inhibition. In this study, we focused on solving the crystal structures of three Llprol mutants (D36S, H38S, and R293S) which behave differently in v-S plots. The D36S and R293S Llprol mutants do not show allosteric behaviour, and the Llprol mutant H38S has allosteric behaviour comparable to the WT enzyme (Hill constant 1.52 and 1.58, respectively). The crystal structures of Llprol variants suggest that the active site of Llprol formed with amino acid residues from both monomers, i.e., located in an interfacial area of dimer. The comparison between the structure models of Llprol indicated that the two monomers in the dimers of Llprol variants have different relative positions among Llprol variants. They showed different interatomic distances between the amino acid residues bridging the two monomers and varied sizes of the solvent-accessible interface areas in each Llprol variant. These observations indicated that Llprol could adapt to different conformational states with distinctive substrate affinities. It is strongly speculated that the domain movements required for productive substrate binding are restrained in allosteric Llprol (WT and H38S). At low substrate concentrations, only one out of the two active sites at the dimer interface could accept substrate; as a result, the asymmetrical activated dimer leads to allosteric behaviour.

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.

Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated ‘omics’ and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches.

Methionine gamma lyase (MGL) is a bacterial and plant enzyme that catalyzes the conversion of methionine in methanthiol, 2-oxobutanoate and ammonia. The enzyme belongs to fold type I of the pyridoxal 5′-dependent family. The catalytic mechanism and the structure of wild type MGL and variants were determined in the presence of the natural substrate as well as of many sulfur-containing derivatives. Structure-function relationship studies were pivotal for MGL exploitation in the treatment of cancer, bacterial infections, and other diseases. MGL administration to cancer cells leads to methionine starvation, thus decreasing cells viability and increasing their vulnerability towards other drugs. In antibiotic therapy, MGL acts by transforming prodrugs in powerful drugs. Numerous strategies have been pursued for the delivering of MGL in vivo to prolong its bioavailability and decrease its immunogenicity. These include conjugation with polyethylene glycol and encapsulation in synthetic or natural vesicles, eventually decorated with tumor targeting molecules, such as the natural phytoestrogens daidzein and genistein. The scientific achievements in studying MGL structure, function and perspective therapeutic applications came from the efforts of many talented scientists, among which late Tatyana Demidkina to whom we dedicate this review.

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long β₄-β₅ loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The β₄-β₅ loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the β₄-β₅ loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant β₂-β_3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site.

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2.