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ACS Nano Jul 2024Proteins are adjustable units from which biomaterials with designed properties can be developed. However, non-native folded states with controlled topologies are hardly...
Proteins are adjustable units from which biomaterials with designed properties can be developed. However, non-native folded states with controlled topologies are hardly accessible in aqueous environments, limiting their prospects as building blocks. Here, we demonstrate the ability of a series of anhydrous deep eutectic solvents (DESs) to precisely control the conformational landscape of proteins. We reveal that systematic variations in the chemical composition of binary and ternary DESs dictate the stabilization of a wide range of conformations, that is, compact globular folds, intermediate folding states, or unfolded chains, as well as controlling their collective behavior. Besides, different conformational states can be visited by simply adjusting the composition of ternary DESs, allowing for the refolding of unfolded states and vice versa. Notably, we show that these intermediates can trigger the formation of supramolecular gels, also known as eutectogels, where their mechanical properties correlate to the folding state of the protein. Given the inherent vulnerability of proteins outside the native fold in aqueous environments, our findings highlight DESs as tailorable solvents capable of stabilizing various non-native conformations on demand through solvent design.
PubMed: 38949563
DOI: 10.1021/acsnano.4c01950 -
The effectiveness of selection in a species affects the direction of amino acid frequency evolution.BioRxiv : the Preprint Server For... Jun 2024Nearly neutral theory predicts that species with higher effective population size ( ) are better able to purge slightly deleterious mutations. We compare evolution in...
UNLABELLED
Nearly neutral theory predicts that species with higher effective population size ( ) are better able to purge slightly deleterious mutations. We compare evolution in high- vs. low- vertebrates to reveal which amino acid frequencies are subject to subtle selective preferences. We take three complementary approaches, two measuring flux and one measuring outcomes. First, we fit non-stationary substitution models of amino acid flux using maximum likelihood, comparing the high- clade of rodents and lagomorphs to its low- sister clade of primates and colugos. Second, we compare evolutionary outcomes across a wider range of vertebrates, via correlations between amino acid frequencies and . Third, we dissect the details of flux in human, chimpanzee, mouse, and rat, as scored by parsimony - this also enables comparison to a historical paper. All three methods agree on which amino acids are preferred under more effective selection. Preferred amino acids tend to be smaller, less costly to synthesize, and to promote intrinsic structural disorder. Parsimony-induced bias in the historical study produces an apparent reduction in structural disorder, perhaps driven by slightly deleterious substitutions. Within highly exchangeable pairs of amino acids, arginine is strongly preferred over lysine, and valine over isoleucine, consistent with more effective selection preferring a marginally larger free energy of folding. These two preferences match differences between thermophiles and mesophilic relatives. These results reveal the biophysical consequences of mutation-selection-drift balance, and demonstrate the utility of nearly neutral theory for understanding protein evolution.
SIGNIFICANCE STATEMENT
According to the nearly neutral theory of molecular evolution, selection is less able to distinguish between similar alleles in species with lower population size. We identify which amino acids are subject to such weak preferences - these tend to be smaller, to be less costly to make, to promote structural disorder of proteins, and to be enriched in thermophiles relative to mesophiles. The latter agrees with theories of marginal protein stability under mutation-selection-drift balance.
PubMed: 38948853
DOI: 10.1101/2023.02.01.526552 -
BioRxiv : the Preprint Server For... Jun 2024Bed bugs are blood-feeders that rapidly proliferate into large indoor infestations. Their bites can cause allergies, secondary infections and psychological stress, among...
Bed bugs are blood-feeders that rapidly proliferate into large indoor infestations. Their bites can cause allergies, secondary infections and psychological stress, among other problems. Although several tactics for their management have been used, bed bugs continue to spread worldwide wherever humans reside. This is mainly due to human-mediated transport and their high resistance to several classes of insecticides. New treatment options with novel modes of action are required for their control. In this study, we evaluated the use of nitisinone (NTBC), an FDA-approved drug, for bed bug control in an insecticide-susceptible (HH) and an insecticide-resistant (CIN) population. Although NTBC was lethal to both populations when administered orally or applied topically in very low doses, we observed a slight but significant resistance in the CIN population. Transcriptomic analysis in both populations indicated that NTBC treatment elicited a broad suppression of genes associated with RNA post-transcriptional modifications, translation, endomembrane system, protein post-translational modifications and protein folding. The CIN population exhibited higher ATP production and xenobiotic detoxification. Feeding studies on a mouse model highlight that NTBC could be used as a control method of bed bugs by host treatment. The results demonstrate that NTBC can be used as a new active ingredient for bed bug control by topical or oral treatment and shed light on the molecular mechanisms of suppressed tyrosine metabolism following NTBC treatment.
PubMed: 38948842
DOI: 10.1101/2024.06.18.599347 -
BioRxiv : the Preprint Server For... Jun 2024Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species...
Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2- based and functional reporter screens in to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting ERGU-1 causes excessive H2O2 and transcriptional gene up- regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behaviors. Both and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. We name this system ER-GUARD, E ndoplasmic R eticulum Gu ardian A egis of R edox D efense. Human and homologs of ERGU-1 can rescue mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. Together, our results reveal an ER-membrane-specific protein machinery and defense-net system ER-GUARD for peroxide detoxification and suggest a previously unknown but conserved pathway for antioxidant defense in animal cells.
PubMed: 38948723
DOI: 10.1101/2024.06.19.599784 -
Endoplasmic reticulum stress and quality control in relation to cisplatin resistance in tumor cells.Frontiers in Pharmacology 2024The endoplasmic reticulum (ER) is a crucial organelle that orchestrates key cellular functions like protein folding and lipid biosynthesis. However, it is highly... (Review)
Review
The endoplasmic reticulum (ER) is a crucial organelle that orchestrates key cellular functions like protein folding and lipid biosynthesis. However, it is highly sensitive to disturbances that lead to ER stress. In response, the unfolded protein response (UPR) activates to restore ER homeostasis, primarily through three sensors: IRE1, ATF6, and PERK. ERAD and autophagy are crucial in mitigating ER stress, yet their dysregulation can lead to the accumulation of misfolded proteins. Cisplatin, a commonly used chemotherapy drug, induces ER stress in tumor cells, activating complex signaling pathways. Resistance to cisplatin stems from reduced drug accumulation, activation of DNA repair, and anti-apoptotic mechanisms. Notably, cisplatin-induced ER stress can dualistically affect tumor cells, promoting either survival or apoptosis, depending on the context. ERAD is crucial for degrading misfolded proteins, whereas autophagy can protect cells from apoptosis or enhance ER stress-induced apoptosis. The complex interaction between ER stress, cisplatin resistance, ERAD, and autophagy opens new avenues for cancer treatment. Understanding these processes could lead to innovative strategies that overcome chemoresistance, potentially improving outcomes of cisplatin-based cancer treatments. This comprehensive review provides a multifaceted perspective on the complex mechanisms of ER stress, cisplatin resistance, and their implications in cancer therapy.
PubMed: 38948460
DOI: 10.3389/fphar.2024.1419468 -
ArXiv Jun 2024We introduce RNA-FrameFlow, the first generative model for 3D RNA backbone design. We build upon SE(3) flow matching for protein backbone generation and establish...
We introduce RNA-FrameFlow, the first generative model for 3D RNA backbone design. We build upon SE(3) flow matching for protein backbone generation and establish protocols for data preparation and evaluation to address unique challenges posed by RNA modeling. We formulate RNA structures as a set of rigid-body frames and associated loss functions which account for larger, more conformationally flexible RNA backbones (13 atoms per nucleotide) vs. proteins (4 atoms per residue). Toward tackling the lack of diversity in 3D RNA datasets, we explore training with structural clustering and cropping augmentations. Additionally, we define a suite of evaluation metrics to measure whether the generated RNA structures are globally self-consistent (via inverse folding followed by forward folding) and locally recover RNA-specific structural descriptors. The most performant version of RNA-FrameFlow generates locally realistic RNA backbones of 40-150 nucleotides, over 40% of which pass our validity criteria as measured by a self-consistency TM-score >= 0.45, at which two RNAs have the same global fold. Open-source code: https://github.com/rish-16/rna-backbone-design.
PubMed: 38947930
DOI: No ID Found -
International Immunopharmacology Jun 2024Heat shock proteins are a widely distributed group of proteins. It is constitutively expressed in almost all organisms and shows little variation throughout evolution....
Tumor-derived Hsp70-CD14 interaction enhances the antitumor potential of cytotoxic T cells by activating tumor-associated macrophages to express CC chemokines and CD40 costimulatory molecules.
Heat shock proteins are a widely distributed group of proteins. It is constitutively expressed in almost all organisms and shows little variation throughout evolution. Previously, HSPs, particularly Hsp70, were recognized as molecular chaperones that aid in the proper three-dimensional folding of newly synthesized polypeptides in cells. Recently, researchers have focused on the potential induction of immune cells, including macrophages, antigen-specific CD8 cytotoxic T cells, and PBMCs. It induces the expression of CC chemokines such as MIP-1α and RANTES, which are responsible for the chemotactic movement and migration of immune cells at the site of infection to neutralize foreign particles in vivo and in vitro in several cell lines but their effect on tumor-associated macrophages is still not known. These cytokines are also known to influence the movement of several immune cells, including CD8 cytotoxic T cells, toward inflammatory sites. Therefore, the effect of tumor-derived autologous Hsp70 on the expression of MIP-lα and RANTES in tumor-associated macrophages (TAMs) was investigated. Our results indicated that Hsp70 treatment-induced MIP-lα and RANTES expression was significantly greater in TAMs than in NMOs. According to the literature, the CC chemokine shares the same receptor, CCR5, as HIV does for their action, and therefore could provide better completion to the virus for ligand binding. Furthermore, Hsp70-preactivated TAMs induced increased IL-2 and IFN-γ expression in T cells during coculture for 48 h and upregulated the antitumor immune response of the host. Therefore, the outcome of our study could be useful for developing a better approach to restricting the growth and progression of tumors.
PubMed: 38944948
DOI: 10.1016/j.intimp.2024.112584 -
Protein Expression and Purification Jun 2024Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently...
Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways.
PubMed: 38944221
DOI: 10.1016/j.pep.2024.106537 -
Cell Reports Jun 2024The unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress through multiple strategies, including reducing protein synthesis, increasing protein...
The unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress through multiple strategies, including reducing protein synthesis, increasing protein folding capabilities, and enhancing misfolded protein degradation. After a multi-omics analysis, we find that signal recognition particle 14 (SRP14), an essential component of the SRP, is markedly reduced in cells undergoing ER stress. Further experiments indicate that SRP14 reduction requires PRKR-like ER kinase (PERK)-mediated eukaryotic translation initiation factor 2α (eIF2α) phosphorylation but is independent of ATF4 or ATF3 transcription factors. The decrease of SRP14 correlates with reduced translocation of fusion proteins and endogenous cathepsin D. Enforced expression of an SRP14 variant with elongation arrest capability prevents the reduced translocation of cathepsin D in stressed cells, whereas an SRP14 mutant without the activity does not. Finally, overexpression of SRP14 augments the UPR and aggravates ER-stress-induced cell death. These data suggest that translocational attenuation mediated by the PERK-SRP14 axis is a protective measure for the UPR to mitigate ER stress.
PubMed: 38943644
DOI: 10.1016/j.celrep.2024.114402 -
Angewandte Chemie (International Ed. in... Jun 2024Peptide folding is a dynamic process driven by non-covalent cross-linking leading to functional nanostructures for essential biochemical activities. However, replicating...
Peptide folding is a dynamic process driven by non-covalent cross-linking leading to functional nanostructures for essential biochemical activities. However, replicating this process in synthetic systems is challenging due to the difficulty in mimicking nature's real-time regulation of non-covalent crosslinking for single-chain polymer folding. Here, we address this by employing anionic dithiol building blocks to create macrocyclic disulfides as non-covalent crosslinkers that adapted to the folding process. Initially, small macrocycles facilitated a low degree folding of a polycation. Then, this preorganized structure catalysed the production of larger macrocycles that enhanced the folding conversely. The self-adaptive synthesis was verified through the encapsulation of an anticancer drug, showing an updated production distribution of non-covalent crosslinkers and maximizing drug-loading efficiency against drug-resistant cancer in vitro. Our research advances the understanding of molecular systems by exploring species evolution via the structural dynamics of polymer folding. Additionally, adaptive synthesis enables controlled, sequential folding of synthetic polymers, with the potential to mimic protein functions.
PubMed: 38943429
DOI: 10.1002/anie.202408670