Protein Science最新文献

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by cognitive decline resulting from neuronal cell death. A key contributor to AD pathology is C99, a membrane-bound β-secretase-cleaved fragment of amyloid precursor protein (APP). C99 plays a central role in generating amyloid-beta (Aβ) isomers, which are directly implicated in disease progression. Understanding its structure and lipid interactions is essential for elucidating its mechanistic role in AD and guiding therapeutic development. C99 has been studied in membrane mimetics such as micelles, bicelles, and reconstituted nanodiscs. Although reconstituted nanodiscs provide a native-like lipid-bilayer environment, the use of detergents prior to reconstitution has been reported to disrupt native folding and lipid-protein interactions. In this study, we successfully isolated and purified C99 along with its associated lipids directly from E. coli cell membranes using a non-ionic pentyl-inulin polymer, avoiding the need for detergents. The purified C99-containing pentyl-inulin nanodiscs were characterized using SDS-PAGE, Western blotting, dynamic light scattering (DLS), ¹H NMR spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS). Notably, we observed SDS-stable oligomers of C99. DLS and ¹H NMR confirmed the presence of large particles composed of pentyl-inulin and E. coli lipids. MALDI-TOF and LC-MS verified the molecular mass and amino acid sequence of C99, respectively. We propose that this detergent-free method for the direct isolation of C99 and native lipids using non-ionic pentyl-inulin may serve as a valuable tool for investigating the C99-secretase complex and for developing compounds aimed at inhibiting the production of amyloid-beta isomers.

The mechanism underlying the pathogenic impact of mutations within intrinsically disordered regions of proteins remains enigmatic, and the mechanisms behind compensatory responses to these perturbations lie within an even deeper veil of obscurity. This study focuses on the compensatory mechanisms of single nucleotide variants within the disordered C-terminal tail of the BTG2 protein, a crucial regulator in cell cycle control. Here, we develop a novel approach by combining molecular dynamics simulations with time-dependent linear response theory to accurately compute the long-distance coupling dynamics between the tail and the structured domain. Using this approach, we reveal how specific mutations can counteract the functional disruptions caused by a known disease-associated mutation, V141M. Our findings demonstrate that the disordered tail regulates critical binding sites allosterically, and a weakening of this modulation may contribute to disease manifestation. However, compensatory mutations restore lost interactions between the disordered region and binding sites, exerting long-distance dynamic control over both critical binding sites and the mutation site 141M. This secondhand allosteric control could be a general mechanism for compensatory mutations to rescue function. These insights not only illuminate the pathogenic mechanisms at play but also offer a framework for identifying potential therapeutic targets in diseases associated with disordered protein regions.

Tau aggregation driven by microtubule-associated protein tau (MAPT) mutations is central to frontotemporal dementia pathology, yet no disease-modifying therapies effectively target mutant tau. Here, we identify purpurin (PUR) and oleocanthal (OLC) as selective inhibitors of mutant tau aggregation using peptide models spanning the R2R3 interface. Biophysical and cellular assays demonstrated that both compounds more effectively inhibit the aggregation of mutant tau peptides compared to wild-type, with PUR preferentially targeting V287I and N279K variants, and OLC showing broader inhibitory activity. Surface plasmon resonance and docking analyses revealed more stable interactions and lower binding free energies with mutant tau, consistent with their enhanced inhibitory effects. Computational studies using monomeric and fibrillar tau structures supported the mutation-specific binding profiles of PUR and OLC. Atomic force microscopy and confocal imaging confirmed reduced fibril formation, while post-transduction treatment assays showed that both compounds significantly suppressed intracellular tau propagation. Additionally, OLC reduced tau phosphorylation and oligomerization in SY5Y-TauP301L-EGFP cells expressing mutant tau. These findings highlight the potential of PUR and OLC as structurally distinct, mutation-targeted inhibitors of tau aggregation and propagation, providing a rationale for their further development as candidate therapeutics for frontotemporal dementia.

Cas12a is one of the most widely used Cas nucleases for genome editing and in vitro diagnosis. A number of engineered Cas12a mutants have been identified with improved activity and stability. However, it remains largely unaddressed how these mutations interact. In a previous study, we used a deep learning model to evolve the stability of Lachnospiraceae bacterium Cas12a (LbCas12a) and obtained about 90 mutants with improved stability. In the present study, we performed a deep analysis of these stabilizing mutations and mutation combinations to understand the stability landscape of LbCas12a. It was found that most of the stabilized mutants had shifted fitness, as characterized by higher trans-cleavage activity at high temperatures but lower activity at the "fit" temperature for the parent protein. These stabilizing mutations were found to have sophisticated epistatic effects. Stabilizing mutation S962K improved protein stability in the context of other stabilizing mutations but by itself exhibited minor improvements. Saturation mutagenesis of S962 had differential effects on the stability of wild-type (WT) LbCas12a and C10L/I976L/C1090D variant, despite similar melting temperatures (T_m) for WT (41.9°C) and C10L/I976L/C1090D (41.1°C). Interestingly, 12 out of 19 amino acid substitutions at S962 reduced the T_m in the context of WT LbCas12a, while 18 out of 19 mutations increased T_m in the C10L/I976L/C1090D variant. We also showed that stabilizing mutations could recover the stability and trans-activity of a destabilized LbCas12a variant. Our findings can facilitate the understanding of LbCas12a natural evolution and provide insights to developing novel engineering strategies for Cas nucleases.

Co- and post-translational modifications can significantly impact the structure, dynamics, and function of proteins. In this study, we investigate how N-terminal acetylation affects misfolding and self-assembly of the enzyme superoxide dismutase 1 (SOD1), implicated in amyotrophic lateral sclerosis (ALS). Studies of protein inclusions in patient samples and animal models have shown that wild-type SOD1 can form amyloid fibrils even when no mutations are found in the sod1 gene. This has identified SOD1 amyloid formation as a possible common denominator of ALS and may suggest that co- and post-translational modifications, like N-terminal acetylation found in human SOD1, can be a factor in disease development. In this work, the impact of N-terminal acetylation of SOD1 on stability and aggregation is characterized. Results show that the structure and thermal stability of the apo state are unaffected by the modification while the amyloid formation rate is significantly enhanced. This is caused by a shortening of the nucleation phase together with an increase of fibril elongation by more than 10-fold upon N-terminal acetylation of SOD1. Collectively, the findings demonstrate how regulation by co- and post-translational modifications can influence protein misfolding and self-assembly.

M2-32 is a non-specific acid phosphatase with a rare ability to function across a broad pH range (3.5-8.5). Analysis using SWISS-PROT Prf Profiles classifies it as a class A acid phosphatase (Z-score: 78.97), sharing 50%-60% sequence similarity with enzymes such as PhoC and PhoN. For detailed characterization, the gene encoding M2-32 was cloned into the pET28(b) vector, overexpressed in Escherichia coli BL21 (DE3), and subsequently purified. Although the monomeric form of M2-32 has a molecular weight of ~28 kDa, size exclusion chromatography, dynamic light scattering, and sedimentation studies revealed a dimeric form in solution. Enzymatic assays using p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, 3'-and 5'-adenosine monophosphate demonstrated robust activity over a pH range of 4.0-8.0 at both 30 and 50°C. Differential scanning fluorimetry indicated an unfolding temperature close to 47°C; however, the enzyme refolded after heat denaturation at 80°C. We have determined the x-ray crystal structure of M2-32 by molecular replacement using an AlphaFold2-guided truncated model, achieving a resolution of 2.2 Å. The protein crystallized as a dimer-of-dimers. Each monomer (residues 38-274) adopts an all-alpha-helical fold composed of 14 helices and two disulfide bonds. Docking studies with adenosine monophosphates, combined with site-directed mutagenesis, identified His174, Arg207, His213, Asp217 as critical catalytic residues, and Tyr136 and Ser172 probably involved in substrate recognition. Mutations at these positions resulted in over 90% loss of enzymatic activity, highlighting their functional significance.

Intrinsically disordered proteins (IDPs) play key roles in various biological processes; they are associated with liquid-liquid phase separation and are targets in disorder-based drug design. Efforts to identify their structural propensities-that can be linked to molecular recognition, malfunction, targeting-still lead to ambiguous results. Secondary structure is routinely assessed by NMR spectroscopy by calculating the secondary chemical shifts (SCSs). Focusing on a given environment in the polypeptide backbone, SCSs highlight the deviation from the "random coil" state. However, the analysis is dependent on which of the numerous random coil chemical shift (RCCS) predictors is applied in the calculations, resulting in an especially pronounced ambiguity for IDPs. To overcome this, we introduce two novel statistical tools that enable the sound identification of structural propensities. We propose the chemical shift discordance ratio (DR) for prefiltering RCCS predictors based on self-consistency. Further on, we introduce the Structural Propensity Identification by t-statistics (SPIT) approach for extracting maximum information from SCS data by using multiple RCCS predictors simultaneously. This way SCS patterns indicating structural propensities can be clearly distinguished from the "noise". The applicability of these methods is demonstrated for four proteins of varying degrees of disorder. Ubiquitin and α-synuclein are used as respective benchmarks for a globular and a disordered protein, while two proline-rich IDPs are included as especially challenging molecules in secondary structure analysis.

The nucleus, as the control center of the eukaryotic cell, is a prime target for therapeutic interventions due to its role in regulating genetic material. Importin-α is critical for successful nuclear import as it recognizes and binds to cargo proteins bearing a classical nuclear localization signal (NLS), which facilitates their transport from the cytoplasm into the nucleus. NLS tagging to 'actively' import therapeutics provides the most effective means of maximizing nuclear localization and therapeutic efficacy. However, traditional NLSs are highly cationic due to the recognition and binding requirements with importin-α. Because of their highly 'super-charged' nature, NLS-tagged therapeutics face significant challenges, including poor pharmacokinetics due to non-specific interactions. In this study, we engineered novel NLS tags with zero net charge to potentially overcome this limitation. Computational modeling and experimental validation revealed that these net-neutral NLSs bind to importin-α with similar modes and energies as their cationic counterpart. High-resolution structural determination and analysis by X-ray crystallography then confirmed their binding modes. Biophysical methods using circular dichroism, microscale thermophoresis, and cellular localization studies demonstrated that these NLSs maintain sufficiently stable complexes and acceptable binding to importin-α and are functional. Additionally, this study revealed that the minor NLS-binding site of importin-α, with its extensive cationic surface area, was particularly suited for interactions with the acidic residues of the net-neutral NLSs. This study provides a foundational understanding of NLS-importin interactions and presents net-neutral NLSs as viable candidates for next-generation NLS-therapeutic development and expands the scope of nuclear-targeting therapies.

Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE-allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen-binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIα transgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%-89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIα transgenic mouse model. In summary, the structures of Can f 1-hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Lectins are proteins or glycoproteins capable of binding specifically and reversibly to carbohydrates, a property that, in itself, gives them great functional versatility in organisms from all kingdoms of nature. A subclass of these proteins, called chimerolectins, is composed of proteins that have at least one lectin domain associated with another functional domain, such as enzymatic domains or modules involved in molecular signaling processes. The emergence of chimerolectins throughout evolution significantly expanded the functional repertoire of lectins, allowing their action to go beyond the interaction with carbohydrates and glycoconjugates. These proteins are involved in the regulation of the immune system in humans and animals, in the defense of plants against pathogens and predators, as well as in the mediation of responses to biotic and abiotic stresses. In addition, they can act as potent lethal toxins or as factors in the infection of several pathogens and are often associated with the manifestation of symptoms of diseases, which makes them therapeutic targets of great interest. Deepening the structural knowledge of these proteins has been essential for understanding their mechanisms of action, in addition to providing solid bases for biotechnological applications and for the rational development of artificial lectins with specific functions. This approach has enabled the creation of chimerolectins with potent antiviral activity, as well as the development of new therapeutic strategies aimed at inducing death in cells of different tumor lineages.