Protein Science最新文献

{"title":"Fine-tuning the yeast GAL10 promoter and growth conditions for efficient recombinant membrane protein production and purification.","authors":"Rebecca Moussa, François Gellé, Sandrine Masscheleyn, Alexandre Pozza, Christel Le Bon, Karine Moncoq, Françoise Bonneté, Bruno Miroux","doi":"10.1002/pro.70125","DOIUrl":"https://doi.org/10.1002/pro.70125","url":null,"abstract":"<p><p>One of the most common issues in producing membrane proteins in heterologous expression systems is the low yield of purified protein. The solubilization efficiency of the recombinant membrane protein from biological membranes is often the limiting step. Here, we study the effects of titration of the GAL10-CYC promoter of Saccharomyces cerevisiae, induction time, and culture media, on the rat mitochondrial uncoupling protein (UCP1) production and solubilization levels. We found that a maximum threshold of solubilized UCP1 (70%) is reached at 0.003% galactose concentration, independently of time, temperature, and detergent-to-protein ratio during solubilization. Supplementation with 0.1% amino acids of the S-lactate medium at induction resumes cell growth and recombinant protein production. The purified UCP1 protein (0.2 mg/L) is homogenous in DDM detergent and active after reconstitution in proteoliposomes. To extend the impact of our findings, we applied the same promoter titration to produce the GFP-AT7B human transporter and found an optimal galactose concentration of 0.0015%. The protein data bank analysis revealed that these galactose concentrations are 300 times lower than usual. We propose a novel strategy for the recombinant production of membrane proteins in the yeast S. cerevisiae, which unlocks the use of this inexpensive eukaryotic host for membrane protein production.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70125"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012841/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Intramolecular epistasis correlates with divergence of specificity in promiscuous and bifunctional NSAR/OSBS enzymes.","authors":"Dat P Truong, Roopa Dharmatti, Dylan Suriadinata, Jamison Huddleston, Rebecca Skouby, Gladys Owusu Addo, Mingzhao Zhu, Anjana Delpe Acharige, Reethu Sankari Bayana, Cristian Davila, Susan C Fults, Frank M Raushel, Kenneth G Hull, Daniel Romo, Margaret E Glasner","doi":"10.1002/pro.70113","DOIUrl":"https://doi.org/10.1002/pro.70113","url":null,"abstract":"<p><p>Understanding the functions and evolution of specificity-determining residues is essential for improving strategies to predict and design enzyme functions. Whether the function of an amino acid residue is retained during evolution depends on intramolecular epistasis, which occurs when the same residue contributes to different phenotypes in different genetic backgrounds. This study examines the relationship between epistasis and functional divergence by investigating a conserved specificity determinant in five homologs from the N-succinylamino acid racemase (NSAR)/o-succinylbenzoate synthase (OSBS) subfamily. NSAR activity originated as a promiscuous (non-biological) activity of an ancestral OSBS. Some extant NSAR/OSBS subfamily enzymes still have OSBS activity as a biological function and NSAR as a promiscuous activity, while some use both OSBS and NSAR activities as biological functions. Others use only NSAR activity as a biological function but can still catalyze the OSBS reaction as a promiscuous activity. Previously, we determined that the conserved residue R266 in Amycolatopsis sp. T-1-60 NSAR contributes to NSAR specificity by enabling K263 to act as a general acid/base catalyst. Here, we show that mutating R266 decreased relative specificity for NSAR activity in four of five NSAR/OSBS subfamily enzymes, as predicted. However, other phenotypes exhibited epistasis related to the pleiotropy of R266, including the proton exchange rate between the catalytic lysines and the substrate, the impact on OSBS activity, and thermostability. The strength of epistasis was associated with functional and evolutionary divergence of NSAR/OSBS enzymes. These results illustrate the benefits of comparing multiple homologs for understanding mechanisms of enzyme specificity.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70113"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006748/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144009029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding the bioanalytical application of β-hydroxybutyrate binding proteins through characterization of their metabolite interactions and site-directed mutagenesis.","authors":"Bryant J Kane, Kyle V Murphy, Koji Sode","doi":"10.1002/pro.70129","DOIUrl":"https://doi.org/10.1002/pro.70129","url":null,"abstract":"<p><p>β-hydroxybutyrate binding proteins (BHBBPs) are a newly identified group of periplasmic solute-binding proteins (SBPs) that interact with β-hydroxybutyrate (BHB), a key physiological metabolite. In this study, we systematically characterized the interaction properties of both previously reported and newly identified BHBBPs, including \"NovoS\" and \"EDC10\" from Gram-negative bacteria. Following recombinant production, we assessed the specificity and affinity of these proteins against a library of 23 different metabolites using a label-free derivative of differential scanning fluorimetry (nanoDSF). Positive interactions were further evaluated for their binding affinity via tryptophan fluorescence spectroscopy, which confirmed D/L-BHB as the preferred ligand for all proteins, with slight enantioselectivity. BHBBPs also exhibited binding to other compounds such as acetoacetate, D/L-α-hydroxybutyrate, L-lactate, and pyruvate, albeit with reduced affinity. These findings expand the classification of BHBBPs, suggesting that similar proteins and associated transporters may be widespread in prokaryotes involved in the carbon cycle of polyhydroxybutyrate. Guided by the crystal structure of the homologous BMA2936 protein, we introduced targeted point mutations in conserved polar residues of the BHBBPs EDC24 and NovoS. It was determined through this experimental pipeline that their affinity towards BHB was reduced by a factor between 25 and 750, shifting their binding constants towards the millimolar range. Collectively, the affinities of both wild-type and mutant proteins span 4 orders of magnitude, from nanomolar to millimolar recognition of BHB. Leveraging the versatility of SBP-based biosensing, these receptors and their wide affinity range could facilitate the development of effective bioanalytical tools for BHB detection in diverse physiological environments.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70129"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012846/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144051067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quaternary stabilization of a GH2 β-galactosidase from the psychrophile A. ikkensis, a flexible and unstable dimeric enzyme.","authors":"Jan S Nowak, Nikoline Kruuse, Helena Ø Rasmussen, Pengfei Tian, Julie Astono, Søren Schultz-Nielsen, Mariane S Thøgersen, Peter Stougaard, Jan Skov Pedersen, Daniel E Otzen","doi":"10.1002/pro.70141","DOIUrl":"https://doi.org/10.1002/pro.70141","url":null,"abstract":"<p><p>Studies of cold-active enzymes may elucidate the basis for low-temperature activity and contribute to their wider application in energy-efficient processes. Here we investigate the cold-active GH2 β-galactosidase from the psychrophilic bacterium Alkalilactibacillus ikkensis (AiLac). AiLac has a specific activity twice as high as its closest structural homolog (the mesophilic Escherichia coli GH2 β-galactosidase) toward the lactose analog ONPG at room temperature and neutral pH, and shows biphasic behavior in Michaelis-Menten plots. AiLac is activated by Mg²⁺ and Na⁺ and is most effective at pH 7.0 and 30°C. However, early unfolding events are observed already at room temperature. Stability studies using intrinsic fluorescence, circular dichroism, and small-angle x-ray scattering (SAXS), combined with activity assays, showed AiLac to be highly sensitive to heat and urea and to be stabilized, but also inhibited, by loss of structural flexibility induced by the osmolyte trehalose. AlphaFold structure prediction combined with SAXS and flow-induced dispersion analysis support a reversible monomer-dimer model, suggesting structural adaptation to cold temperatures on a quaternary level. The low amount of dimeric buried surface area, high flexibility, and remarkably low chemical and thermal stability present an extreme example of cold adaptation promoted by high levels of solvent interactions. To investigate the relationship between evolution and oligomerization, we trained a generative deep learning model to successfully engineer functional variants that form stabilized dimers and tetramers by introducing high evolutionary fitness mutations at the interface, demonstrating an efficient way to explore the local sequence fitness landscape to modulate the equilibrium of oligomerization.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70141"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12023411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144041228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Weak, specific chemical interactions dictate barnase stability in diverse cellular environments.","authors":"Ume Tahir, Caitlin M Davis","doi":"10.1002/pro.70128","DOIUrl":"https://doi.org/10.1002/pro.70128","url":null,"abstract":"<p><p>It is well-established that in vitro measurements do not reflect protein behaviors in-cell, where macromolecular crowding and chemical interactions modulate protein stability and kinetics. Recent work suggests that peptides and small proteins experience the cellular environment differently from larger proteins, as their small sizes leave them primarily susceptible to chemical interactions. Here, we investigate this principle in diverse cellular environments, different intracellular compartments and host organisms. Our small protein folding model is barnase, a bacterial ribonuclease that has been extensively characterized in vitro. Using fast relaxation imaging, we find that FRET-labeled barnase is stabilized in the cytoplasm and destabilized in the nucleus of U2-OS cells. These trends could not be reproduced in vitro by Ficoll and M-PER™, which mimic macromolecular crowding and non-specific chemical interactions, respectively. Instead, in-cell trends were best replicated by cytoplasmic and nuclear lysates, indicating that weak specific interactions with proteins in either compartment are responsible for the in-cell observations. Interestingly, in the cytoplasm barnase's unfolded state is unstable and prone to aggregation, while in the nucleus a stable unfolded state exists prior to aggregation. In the more biologically relevant environment of bacterial cells, barnase folding resembled that in the nucleus, but with no aggregation at higher temperatures. These findings show that protein interactions are evolved for their native environment, which highlights the importance of studying and designing proteins in situ.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70128"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006822/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural insights into the interaction between testis-specific Y-encoded-like protein 5 and ubiquitin-specific protease 7.","authors":"Marine Ancia, Khadija Wahni, Joudy Chakrowf, Asia El Aakchioui, Eloïse Claude, Guillaume de Lhoneux, Maxime Liberelle, Steven Janvier, Ekaterina Baranova, Julia Malo Pueyo, Ariana Jijon Vergara, Nicolas Papadopoulos, Clémence Balty, Jérôme Dejeu, Anabelle Decottignies, Joris Messens, Raphaël Frédérick","doi":"10.1002/pro.70116","DOIUrl":"https://doi.org/10.1002/pro.70116","url":null,"abstract":"<p><p>The Alternative Lengthening of Telomeres (ALT) mechanism enables telomere maintenance, contributing to the immortality of certain cancer cells. Disrupting the interaction between testis-specific Y-encoded-like protein 5 (TSPYL5) and ubiquitin-specific protease 7 (USP7) has emerged as a promising strategy to target ALT-dependent cancers. While the N-terminal MATH domain of USP7 mediates the protein interaction, the regions of TSPYL5 involved in binding remain unclear. Here, we present a structural analysis of the TSPYL5-USP7 interaction to guide targeted therapeutic strategies. We showed that TSPYL5 is intrinsically disordered, with an unfolded N-terminal region and partial structure in the C-terminal half. In vitro, recombinantly expressed TSPYL5 binds USP7 with nanomolar affinity and is prone to C-terminal truncation. However, the truncated form retained a similar binding affinity for USP7, suggesting the primary interaction site resides in the N-terminal region of TSPYL5. We identified three key binding hotspots within TSPYL5: residues 65-97, residues 210-262, and residues 368-388. Moreover, TSPYL5 forms trimers that further assemble into hexamers. This study provides the first structural and quantitative analysis of the TSPYL5-USP7 interaction, highlighting these three binding sites. These findings lay the groundwork for the development of novel inhibitors targeting ALT-dependent cancers.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70116"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012732/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144046049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An evolutionarily conserved tryptophan cage promotes folding of the extended RNA recognition motif in the hnRNPR-like protein family.","authors":"Ernest S Atsrim, Catherine D Eichhorn","doi":"10.1002/pro.70127","DOIUrl":"https://doi.org/10.1002/pro.70127","url":null,"abstract":"<p><p>The heterogeneous nuclear ribonucleoprotein (hnRNP) R-like family is a class of RNA binding proteins in the hnRNP superfamily with diverse functions in RNA processing. Here, we present the 1.90 Å X-ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N-terminal extension (N_ext) motif that docks against the RRM and extends the β-sheet surface. The adjoining loop is structured and forms a tryptophan cage motif to position the N_ext motif for docking to the RRM. Combining mutagenesis, solution NMR spectroscopy, and thermal denaturation studies, we evaluate the importance of residues in the N_ext-RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, conformational ordering, and thermal stability. Consistent with their importance, mutations in the N_ext-RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR-like proteins reveals conserved features in the eRRM.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70127"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006756/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144042150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The structural basis for the broad aldehyde specificity of the aminoaldehyde dehydrogenase PauC from the human pathogen Pseudomonas aeruginosa.","authors":"Yudy V Cardona-Cardona, Lilian González-Segura, Rogelio Rodríguez-Sotres, Javier Andrés Juárez-Díaz, Carlos Mújica-Jiménez, Ignacio Regla, Manuel López-Ortiz, Rosario A Muñoz-Clares","doi":"10.1002/pro.70124","DOIUrl":"https://doi.org/10.1002/pro.70124","url":null,"abstract":"<p><p>Despite significant differences in size and formal charge, the aldehyde dehydrogenase PaPauC (PA5312) from Pseudomonas aeruginosa PAO1 efficiently catalyzes the NAD⁺-dependent oxidation of the aminoaldehydes formed in polyamines degradation. We report here that PaPauC also oxidizes 4-guanidinebutyraldehyde, formed in one arginine degradation pathway, trimethylaminobutyraldehyde, of unknown metabolic origin, and indole-3-acetaldehyde, a precursor of the plant growth-promoting hormone indoleacetic acid. PaPauC has been proposed as a potential target for combating P. aeruginosa. However, understanding its structure-function relationships, crucial for developing specific inhibitors, is lacking. Using X-ray crystallography, we identified the structural characteristics that determine PaPauC broad aldehyde specificity: a spacious aldehyde-entrance tunnel and six active-site residues. Docking simulations, site-directed mutagenesis, and kinetic analyses support the interactions of Lys479 with glutamylated aminoaldehydes; Phe169, Trp176, and Phe467 with amino and guanidinium groups through cation-π interactions and with the indole group via NH-π and CH-π interactions; Asp459 with amino and indole groups; and Thr303 with amide and guanidinium groups. Exploiting the distinctive structural features of the PaPauC active site could aid in developing specific inhibitors to combat P. aeruginosa infections in humans and animals, as well as in preventing its colonization of plants, which are abundant P. aeruginosa reservoirs and, therefore, a significant source of human infections.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70124"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006823/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144050712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure and inhibition of diaminopimelic acid epimerase by slow-binding α-methyl amino acids.","authors":"Tess Lamer, Pu Chen, Karizza Catenza, Ilia Perov, Bethan L, Yu-Ting Hsiao, Tayla J Van Oers, M Joanne Lemieux, John C Vederas","doi":"10.1002/pro.70139","DOIUrl":"https://doi.org/10.1002/pro.70139","url":null,"abstract":"<p><p>Cofactor-independent racemases and epimerases produce D-amino acids from their L-isomers for a variety of biological processes. These enzymes operate via an unusual mechanism that relies on an active site cysteine thiolate (pK_a ~ 8.5) to deprotonate an amino acid α-carbon (pK_a ~ 29) and are of interest not only because of their biocatalytic potential for D-amino acid production, but also because many play key roles in biology and are antibiotic targets. However, obtaining crystal structures of these enzymes, especially in their closed, substrate- or inhibitor-bound conformations, is difficult. In this work, we characterized diaminopimelic acid (DAP) epimerase from the cyanobacterium Anabaena. DAP epimerase has long been of interest as an antibiotic target as it converts L,L-DAP to D,L-DAP for lysine and peptidoglycan biosynthesis. We solved three crystal structures of this enzyme in its closed, inhibitor-bound conformation, up to a resolution of 1.5 Å. Two structures show the enzyme covalently bound through its catalytic cysteine residues to previously reported aziridine-based inhibitors. One structure unexpectedly shows the enzyme bound to a different compound, D,L-α-methylDAP, presumably produced as a synthetic byproduct. Stereoselective synthesis of L,L- and D,L-α-methylDAP followed by inhibition assays shows that these compounds are slow-binding inhibitors of DAP epimerase. α-MethylDAP inhibitors provide a more accessible alternative to aziridine-based inhibitors to obtain crystal structures of DAP epimerase in its closed conformation. Comparisons of bacterial, cyanobacterial, and plant DAP epimerases provided here offer new insights into functional and structural differences between these enzymes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70139"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12039745/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The role of phospholipid saturation and composition in α-synuclein aggregation and toxicity: A dual in vitro and in vivo approach.","authors":"Aidan P Holman, Tianyi Dou, Mikhail Matveyenka, Kiryl Zhaliazka, Anjni Patel, Avery Maalouf, Ragd Elsaigh, Dmitry Kurouski","doi":"10.1002/pro.70121","DOIUrl":"https://doi.org/10.1002/pro.70121","url":null,"abstract":"<p><p>Parkinson's disease is characterized by a progressive accumulation of α-synuclein (α-syn) aggregates in Lewy bodies, extracellular deposits found in the midbrain, hypothalamus, and thalamus. The rate of α-syn aggregation, as well as the secondary structure of α-syn oligomers and fibrils, can be uniquely altered by lipids. However, the role of saturation of fatty acids (FAs) in such lipids in the aggregation properties of α-syn remains unclear. In this study, we investigated the effect of saturation of FAs in phosphatidylcholine (PC) and cardiolipin (CL), as well as a mixture of these phospholipids on the rate of α-syn aggregation. We found that although saturation plays very little if any role in the rate of protein aggregation and morphology of α-syn aggregates, it determined the secondary structure of α-syn oligomers and fibrils. Furthermore, we found that aggregates formed in the presence of both saturated and unsaturated PC and CL, as well as mixtures of these phospholipids, exert significantly higher cell toxicity compared to the protein aggregates formed in the lipid-free environment. To extend these findings, we conducted in vivo studies using C. elegans, where we assessed the effect of lipid-modified α-syn aggregates on organismal survival and neurotoxicity. Our results suggest that the saturation of FAs in phospholipids present in the plasma and mitochondrial membranes can be a key determinant of the secondary structure and, consequently, the toxicity of α-syn oligomers and fibrils. These findings provide new insights into the role of lipids in Parkinson's disease pathogenesis and highlight potential targets for therapeutic intervention.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 5","pages":"e70121"},"PeriodicalIF":4.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006753/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144022077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}