Native Complex Research Articles

Engineering Native Biological Complexity from the Inside-out and Outside-in.

Focusing on engineering control over cell function and fate, this article examines the critical balance of 'outside-in' and 'inside-ouť signaling in tissue development and regeneration. It highlights emerging strategies to manipulate these interactions, including biomaterial design and synthetic biology to influence this delicate equilibrium and fine tune cellular responses.

Novel pathomechanisms of impairment of native tear protein complexes in aqueous deficient dry eye syndrome

Aims/Purpose: Dry eye syndrome (DES) is a common yet deplorable disease of the ocular surface with high global prevalence. The pathomechanisms underlying the impairment of the native tear protein complexes remain unexplored. This study established a quantitative proteomics approach to characterize the novel protein complexes and unravel their functional roles in tears of DES patients.Methods: Tear samples were collected using Schirmer's strips from aqueous‐deficient DES (DRY, n = 16) and healthy (CTRL, n = 16) subjects following comprehensive DES clinical examinations. The native tear protein complex profiles were elucidated employing a customized blue native gel electrophoretic (BN‐PAGE) combined with mass spectrometry (MS)‐based proteomics strategy. In‐silico bioinformatics tools were employed for gene annotation analysis of the complexes.Results: Five major native protein complexes designated as complex I (cI, 800 kDa), II (cII, 400 kDa), III (cIII, 240 kDa), IV (cIV, 140 kDa) and V (cV, 66 kDa) were characterized for the first time in human tears. Interestingly, a cluster of immune‐related proteins identified in cI (e.g., IGHA1) were annotated to complement pathways and was found to be significantly increased in abundance in DRY vs. CTRL (p = 8.2 × 10−3). Likewise, increased abundance of another cluster of proteins annotated to inflammatory response (e.g. S100A9) in DRY (p = 1.7 × 10−2) was associated to cIII. On the contrary, cII was largely composed of lacrimal‐specific proteins (e.g., LYZ), which was found to be decreased in abundance in DRY (p = 2.4 × 10−2). Similarly, decrement of antimicrobial proteins in cIV (e.g., LACRT) was observed in DRY (p = 1.7 × 10−2). The expression of a cluster of carrier proteins involved in protein–protein interactions (e.g., ALB) was annotated in cV, which was increased in DRY (p = 4.4 × 10−3). Noteworthy, 35 new DES markers were identified with this methodological approach.Conclusions: Collectively, a tear‐customized BN‐PAGE‐MS analysis was instrumental to decipher the multifaceted biological functions of novel native protein complexes in human tears. For the first time, the intricate molecular mechanistic changes underlying these protein complexes in DES were unravelled, which could be instrumental for potential development of therapeutic strategies in ameliorating DES.

Purification of Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography.

Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).

Use of thistle seeds of modified composition in chocolate mass technology

The object of the study is the analytical values of lipids of milk thistle seeds of a modified composition, namely acid and peroxide values, as well as the induction period of lipids of chocolate mass with the addition of milk thistle seeds. The paper substantiates rational parameters of thistle seed processing for the inactivation of lipases and lipoxygenases. Approximate dependences of the acid and peroxide values of thistle seed lipids on the pH of the wetting solution and the wetting degree of seeds were obtained. This makes it possible to substantiate such rational processing parameters of thistle seeds that inhibit the accumulation of free fatty acids and primary oxidation products in seeds during storage. An increase in the induction period of the oxidation of the lipid component of the chocolate mass with the use of thistle seeds of a modified composition was proven, which is 2.5 times higher than the induction period of the chocolate mass sample with thistle seeds with a native enzyme complex. The research results make it possible to develop a technology of healthy chocolate mass using thistle seeds of a modified composition, the lipid component of such mass is stable to oxidation and hydrolysis. The data obtained in the work are explained by an increase in the ability to inactivate lipolytic and lipoxygenase enzymes of milk thistle at high humidity in an acidic environment under the influence of microwave radiation. The advantage of the obtained results is the possibility of not violating the integrity of thistle seeds during the inactivation of the enzyme complex, which allows extending the shelf life of this raw material. An applied aspect of using the scientific result is the possibility of expanding the range of healthy chocolate masses with the use of thistle seeds

Atomistic modelling and NMR studies reveal that gallium can target the ferric PQS uptake system in P. aeruginosa biofilms.

Intravenous gallium nitrate therapy is a novel therapeutic strategy deployed to combat chronic Pseudomonas aeruginosa biofilm infections in the lungs of cystic fibrosis (CF) patients by interfering with iron (Fe3+) uptake. The therapy is a source of Ga3+, which competes with Fe3+ for siderophore binding, subsequently disrupting iron metabolism and inhibiting biofilm proliferation in vivo. It was recently demonstrated that the Pseudomonas quinolone signal (PQS) can chelate Fe3+ to assist in bacterial iron uptake. However, it is unknown whether exogenous gallium also targets [Fe(PQS)3] uptake, which, in turn, would extend the mechanism of gallium therapy beyond siderophore competition, potentially supporting use of the therapy against P. aeruginosa mutants deficient in siderophore uptake proteins. To that end, the thermodynamic feasibility of iron-for-gallium cation exchange into [Fe(PQS)3] was evaluated using quantum chemical density functional theory (DFT) modelling and verified experimentally using 1H nuclear magnetic resonance (NMR). We demonstrate here that Ga3+ can strongly bind to three PQS molecules and, furthermore, displace and substitute Fe3+ from the native chelate pocket within PQS complexes, through a Trojan horse mechanism, retaining the key structural features present within the native ferric complex. As such, [Fe(PQS)3] complexes, in addition to ferric-siderophore complexes, represent another target for gallium therapy.

Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present challenges to study using conventional structural biology techniques. Here we develop a native nanoproteomics strategy for the enrichment and subsequent native top-down mass spectrometry (nTDMS) analysis of endogenous cardiac troponin (cTn) complex directly from human heart tissue. The cTn complex is enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complex, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the cTn complex, localizes Ca2+ binding domains, defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a paradigm for structural characterization of endogenous native protein complexes.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Functional Coupling of Biohybrid Photosynthetic Antennae and Reaction Center Complexes: Quantitative Comparison with Native Antennae.

Light-harvesting (LH) complexes in photosynthetic organisms absorb photons within limited wavelength ranges over a broad solar spectrum. Extension of the LH wavelength has been realized by attaching artificial fluorophores to LH complexes (biohybrid LH complexes) for complementing the limited-wavelength regions. However, how efficiently such fluorophores in biohybrid LH complexes function to drive the photocatalytic reaction center (RC) has not been quantitatively evaluated, specifically in comparison with native LH antenna complexes. In this study, we prepared various biohybrid LH1-RC complexes (from Rhodopseudomonas palustris), to quantitatively evaluate the LH activity of the attached external chromophores through a photocurrent generation reaction by LH1-RC on an electrode. For a direct comparison of the LH activity among the LH chromophores that were examined, we introduced the k1 term, which represents the extent of the functional coupling of LH and the photochemical reactions in the RC. We determined that the hydrophobic fluorophore ATTO647N attached to LH1 possesses the highest LH activity among the examined hydrophilic fluorophores such as Alexa647, and its activity is comparable to that of native LH1(-RC). The LH activity of LH2 (from Rhodoblastus acidophilus strain 10050) and its biohybrid LH2s were examined for the comprehensive assessment of their LH activity.

An Inducible ESCRT-III Inhibition Tool to Control HIV-1 Budding.

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

Antibacterial Activity of Rainbow Trout Plasma: In Vitro Assays and Proteomic Analysis.

The objective of this study was to investigate the bactericidal activity of blood plasma from cultured rainbow trout obtained from two different fish farms. Plasma from trout naturally infected with the bacterial pathogen Flavobacterium psychrophilum was found to inhibit the growth of Aeromonas hydrophila in vitro. Incubation of A. hydrophila in bacteriostatic trout plasma resulted in agglutination and growth retardation, without causing massive damage to the cell membrane. The proteome of the plasma with high antimicrobial activity revealed an abundance of high-density apolipoproteins, some isoforms of immunoglobulins, complement components C1q and C4, coagulation factors, lectins, periostin, and hemoglobin. Analysis of trout proteins retained on A. hydrophila cells revealed the presence of fish immunoglobulins, lectins, and complement components on bacteria whose growth was inhibited, although the native membrane attack complex of immunised trout plasma did not assemble effectively, resulting in a weak bactericidal effect. Furthermore, this study examined the bacterial response to trout plasma and suggested that the protein synthesis pathway was the target of antimicrobial proteins from fish blood. Taken together, these findings illustrate the advantages of the affinity approach for understanding the role of plasma proteins in host defence against pathogens.

Exploring natural products library to identify potential inhibitors targeting isoniazid-resistant tuberculosis

Mycobacterium tuberculosis (MTB) causing tuberculosis (TB) infection is a leading source of illness and death in developing nations, and the emergence of drug-resistant TB remains a significant global threat and a challenge in treating the disease. Mutations in the inhA and katG genes are connected to the principal molecular mechanism of isoniazid (INH) resistance, and continuous treatment of INH for more than a decade led to the evolution of INH resistant-TB (inhR-TB). Structure-based drug discovery approaches on traditional natural compounds are the contemporary source to identify significant lead molecules. This work focuses on discovering effective small compounds from natural compound libraries and applying pharmacophore-based virtual screening to filter out the molecules. The best-identified hit complexes were used for molecular dynamics simulations (MDS) to observe their stability and compactness. A three-dimensional e-pharmacophore hypothesis and screening generated 62 hits based on phase fitness scores from the pharmacophore-based virtual screening. Molecular docking experiments in Maestro’s GLIDE module indicated that ZINC000002383126 and ASN22022 may be potential inhibitors of inhA and katG (native, inhA mutants S94A, Y158A, Y158F and Y158S and D137S, Y229F, S315T, W321F, and R418L mutants of katG). In addition, MDS analysis indicated that the native and mutant docked complexes of inhA and katG had good stability and remained compact in the binding pocket of the targets. In vitro studies can further validate the compounds that can act as INH competitive inhibitors. Communicated by Ramaswamy H. Sarma

Elucidating the dynamic remodelling of Escherichia coli interactome in different growth conditions using multiplex co-fractionation MS (mCF-MS).

Most proteins function by forming complexes within a dynamic interconnected network that underlies various biological mechanisms. To systematically investigate such interactomes, high-throughput techniques, including CF-MS, have been developed to capture, identify, and quantify protein-protein interactions (PPIs) on a large scale. Compared to other techniques, CF-MS allows the global identification and quantification of native protein complexes in one setting, without genetic manipulation. Furthermore, quantitative CF-MS can potentially elucidate the distribution of a protein in multiple co-elution features, informing the stoichiometries and dynamics of a target protein complex. In this issue, Youssef etal. (Proteomics 2023, 00, e2200404) combined multiplex CF-MS and a new algorithm to study the dynamics of the PPI network for Escherichia coli grown under ten different conditions. Although the results demonstrated that most proteins remained stable, the authors were able to detect disrupted interactions that were growth condition specific. Further bioinformatics analyses also revealed the biophysical properties and structural patterns that govern such a response.

Enhancing Zinc Uptake through Dual-Modification of Cicer arietinum Protein

Zinc is crucial for physiological processes; however, deficiency persists globally. Binding zinc to plant proteins enhances absorption, minimizing toxicity risks and offering a potential solution for deficiency. Mineral binding efficiency of the unmodified protein is limited; hence, dual modification (succinylation and ultrasonication) is potentially used to achieve higher binding efficiency. Enhancing zinc uptake is crucial for cellular health due to its vital roles in various biological processes including enzymatic activity, DNA repair, immune function, antioxidant defense, hormone regulation, brain function, signaling, growth, gene expression, and reproduction. Therefore, this research aimed to develop a chickpea protein-zinc complex and to evaluate the influence of dual modification on their physiochemical, bioavailability, and cellular mineral uptake attributes. Succinylation exhibited significant improvements in the water-holding capacity by 28.73%, oil-holding capacity by 34.09%, and solubility by 5.46% of the chickpea protein-zinc complex as compared to the native complex. Mineral bioavailability increased by 8.32%, and there were notable increases in cellular uptake of zinc by 2.10%, retention by 5.80%, and transport by 3.96%, respectively. Furthermore, the dual modification approach resulted in a notable decrease in the particle size of the chickpea protein-zinc complex, with a substantial reduction of 73.25% and an increased zeta potential value of −21 mV compared to the succinylated complex. As well, dual modification concurrently led to a substantial decline of 48.04% in the sulfhydryl (SH) content, coupled with a marked increase of 21.92% in the surface hydrophobicity. In addition, zinc bioavailability, cellular uptake, retention, and transport were further enhanced by 1.89, 3.34, and 4.8% through dual modification. Our findings highlight that the dual modification of the chickpea protein-zinc complex shows a promising strategy for enhancing the techno-functional characteristics, bioavailability, and cellular uptake of zinc, which could be a better platform for developing vegan foods.

Topological links in predicted protein complex structures reveal limitations of AlphaFold

AlphaFold is making great progress in protein structure prediction, not only for single-chain proteins but also for multi-chain protein complexes. When using AlphaFold-Multimer to predict protein‒protein complexes, we observed some unusual structures in which chains are looped around each other to form topologically intertwining links at the interface. Based on physical principles, such topological links should generally not exist in native protein complex structures unless covalent modifications of residues are involved. Although it is well known and has been well studied that protein structures may have topologically complex shapes such as knots and links, existing methods are hampered by the chain closure problem and show poor performance in identifying topologically linked structures in protein‒protein complexes. Therefore, we address the chain closure problem by using sliding windows from a local perspective and propose an algorithm to measure the topological–geometric features that can be used to identify topologically linked structures. An application of the method to AlphaFold-Multimer-predicted protein complex structures finds that approximately 1.72% of the predicted structures contain topological links. The method presented in this work will facilitate the computational study of protein‒protein interactions and help further improve the structural prediction of multi-chain protein complexes.

Fish know no borders—Implications of the genetic structure and mixed‐stock composition to cross‐border management of adfluvial brown trout

AbstractEffective management of migrating fish stocks requires an understanding of population structure and each stock's contribution to the fishery. Water bodies spanning across national borders pose a particular challenge. We investigated the genetic structure of 13 adfluvial brown trout populations from natal rivers and conducted a genetic mixed‐stock analysis of brown trout from the cross‐border Lake Pyaozero Basin in eastern Fennoscandia, using 16 microsatellite loci. Our analysis revealed a clear genetic structure of three distinct geographic groups, consisting of river‐specific populations. The Western and Southern groups, which included 11 rivers, contributed over 90% to the mixed stock in the lacustrine rearing area. This mixed stock of several river‐specific subpopulations renders poorly managed fishing in the area a significant threat to the entire native adfluvial brown trout population complex.

Unlocking the Mysteries of Chromatin Biology through Functional Epigenomics.

In the ever-evolving field of functional genomics, CRISPR-based screening technologies have become pivotal tools for elucidating gene function across various cell types. A recent study by Gilan and colleagues advances this technological frontier by introducing CRISPR-ChIP, a platform designed to investigate the complex dynamics of epigenetic regulation of chromatin. In proof-of-concept experiments, the authors demonstrate the potential of this tool to identify key molecular regulators of two major histone modifications associated with active transcription, H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 79 dimethylation (H3K79me2). They further unveiled a previously unknown functional partitioning of the H3K79-specific methyltransferase DOT1L into an oncogenic complex with MLL-AF9 and a native complex with MLLT10, which cooperatively regulate Mixed Lineage Leukemia fusion protein (MLL-FP) target gene expression. This novel epigenomic approach integrates high-throughput CRISPR screening with ChIP-based direct readout of chromatin modifications in situ, offering a powerful tool to investigate the epigenetic regulatory layers across a diverse spectrum of biological processes and disease states.

Native defects association enabled room-temperature p-type conductivity in β-Ga2O3

The room temperature hole conductivity of the ultra-wide bandgap semiconductor β-Ga2O3 is a pre-requisite for developing the next-generation electronic and optoelectronic devices based on this oxide. In this work, high-quality p-type β-Ga2O3 thin films grown on r-plane sapphire substrate by metalorganic chemical vapor deposition (MOCVD) exhibit ρ = 5 × 104 Ω·cm resistivity at room temperature. A low activation energy of conductivity as Ea2 = 170 ± 2 meV was determined, associated to the VO++−VGa−native acceptor defect complex. Further, taking advantage of cation (Zn) doping, the conductivity of Ga2O3:Zn film was remarkably increased by three orders of magnitude, showing a long-time stable room-temperature hole conductivity with the conductivity activation energy of around 86 meV. VO++−ZnGa− defect complex has been proposed as a corresponding shallow acceptor.

Molecular analysis of the type III interferon complex and its applications in protein engineering

Type III interferons (IFNλs) are cytokines with critical roles in the immune system and are attractive therapeutic candidates due to their tissue-specific activity. Despite entering several clinical trials, results have demonstrated limited efficacy and potency, partially attributed to low-affinity protein-protein interactions (PPIs) responsible for receptor complex formation. Subsequently, structural studies of the native IFNλ signaling complexes remain inaccessible. While protein engineering can overcome affinity limitations, tools to investigate low-affinity systems like these remain limited. To provide insights into previous efforts to strengthen the PPIs within this complex, we perform a molecular analysis of the extracellular ternary complexes of IFNλ3 using both computational and experimental approaches. We first use molecular simulations and modeling to quantify differences in PPIs and residue strain fluctuations, generate detailed free energy landscapes, and reveal structural differences between an engineered, high-affinity complex, and a model of the wild-type, low-affinity complex. This analysis illuminates distinct behaviors of these ligands, yielding mechanistic insights into IFNλ complex formation. We then apply these computational techniques in protein engineering and design by utilizing simulation data to identify hotspots of interaction to rationally engineer the native cytokine-receptor complex for increased stability. These simulations are then validated by experimental techniques, showing that a single mutation at a computationally predicted site of interaction between the two receptors increases PPIs and improves complex formation for all IFNλs. This study highlights the power of molecular dynamics simulations for protein engineering and design as applied to the IFNλ family but also presents a potential tool for analysis and engineering of other systems with low-affinity PPIs.

Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging

—Proteasomes are key components of the ubiquitin–proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit β5; increased expression of genes encoding immune proteasome subunits β5i and β1i. In tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the β5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Elucidation of binding mechanism, affinity, and complex structure between mWT1 tumor-associated antigen peptide and HLA-A*24:02.

We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved x-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the major histocompatibility complex (MHC) molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine.