Tertiary Structure Research Articles

Tuning ProteinMPNN to reduce protein visibility via MHC Class I through direct preference optimization.

ProteinMPNN is widely used in protein design workflows due to its ability to identify amino acid sequences that fold into specific 3D protein structures. In our work, we adjust ProteinMPNN to design proteins for a given 3D protein structure with reduced immune-visibility to cytotoxic T lymphocytes that recognize proteins via the MHC-I pathway. To achieve this, we developed a novel framework that integrates Direct Preference Optimization (DPO)-a tuning method originally designed for large language models-with MHC-I peptide presentation predictions. This approach fosters the generation of designs with fewer MHC-I epitopes while preserving the protein's original structure. Our results demonstrate that DPO effectively reduces MHC-I visibility without compromising the structural integrity of the proteins. Source code: https://github.com/hcgasser/CAPE_MPNN.

Excellent quality acquisition of myofibrillar protein in red shrimp (Solenocera crassicornis) based on regulating the oxidation degree of atmospheric cold plasma treatment.

Myofibrillar protein (MP) is essential for the texture and taste of shrimp surimi products. The reactive oxygen/nitrogen species (ROS/RNS) produced by atmospheric cold plasma (ACP) might cause oxidative modification of MP. In this study, the effect of different ACP treatment times on the properties of red shrimp MP was investigated in detail. The mild oxidation induced by ACP treatment for 1 min (ACP-1 min) promoted the unfolding and refolding of the MP structure, which was manifested as a transition from α-helix to β-sheets. Compared with other groups, more hydrogen bonds and hydrophobic interactions in the ACP-1 min group might enhance the interactions between the myosin heavy chain and actin, which was conducive to the formation of a regular and dense three-dimensional network structure. Person correlation analysis revealed that ROS/RNS mediated the changes in the secondary and tertiary structure of MP. In addition, ACP-1 min has high Ca2+-ATPase activity, which reflected that MP maintained structural integrity. While excessive oxidation in the ACP treatments for 3 min and 5 min reduced MP robustness. The stable internal structure in ACP-1 min group gave the MP excellent texture profile (1.79 mJ of adhesiveness and 0.89 mm of springiness) and rheological characteristics (0.373 MPa of storage modulus). Overall, the excellent quality of MP could be obtained by regulating the oxidation degree of ACP, which would provide valuable information for the in-depth and efficient processing of shrimp products. © 2024 Society of Chemical Industry.

Intertumoral heterogeneity of the immune microenvironment in high grade canine mast cell tumors

BackgroundCanine cutaneous mast cell tumors (MCTs) are a common, yet clinically challenging tumor type given their variable biological behavior. Although patients with low grade MCTs can often be effectively managed with surgery alone, most dogs with high grade MCTs succumb to their disease despite multimodal therapy. An improved understanding of the immune tumor microenvironment (TME) may help identify novel prognostic and therapeutic targets.MethodsIn this study, we interrogated the immune transcriptional profiles of the TME in low and high grade MCTs, and quantified intratumoral T cells. Twelve client-owned dogs with MCTs (6 Kiupel low grade with clinically benign behavior and 6 Kiupel high grade with clinically aggressive behavior) that underwent curative-intent surgery were selected. Tumor grade was confirmed by a single veterinary pathologist. RNA was extracted from all tumors followed by immune transcriptional profiling utilizing the NanoString Canine IO panel and analysis using the ROSALIND platform. T cell density was determined by immunohistochemical staining for CD3 and quantified using ImageScope software (Leica Biosystems) following digital slide capture. Lymphocytic infiltrate was further characterized in the TME of one high grade MCT using co-immunofluorescence.ResultsImmune transcriptional profiling identified 9 differentially expressed genes between low and high grade MCTs (p-adj < 0.05). Programmed cell death protein 1 (PDCD1) and inducible T-cell costimulator ligand (ICOSLG) gene expression were significantly higher in a subset of high grade MCTs. ICOSLG expression positively correlated with T cell score (rs = 0.6434, p = 0.0278). Although the T cell density was not significantly different between low (mean of 76.42 CD3 + /mm2, SD 12 CD3 + /mm2) and high grade MCTs (mean of 129.1 CD3 + /mm2, SD 96.06 CD3 + /mm2), greater variation of T cell densities was observed across high grade MCTs compared to low grade (p = 0.0059). Immunofluorescence of one high grade MCT with marked T cell infiltration revealed organized aggregates of T and B cells consistent with tertiary lymphoid structures (TLS).ConclusionsOur data revealed significant differences in the immune TME of low and high grade MCTs and provides rationale to further investigate potential prognostic and therapeutic roles of immune checkpoints in canine MCTs.

In-Silico and Computational Active Site-Driven Design and Characterization of Beta-NGF-Aβ Fusion Protein as a Novel and Targeted Alzheimer’s Disease Therapy

The targeted delivery of therapeutic chimeric molecules significantly enhances pharmacological efficiency by triggering strong cellular uptake and prolonging the duration of systemic circulation. Purposeful molecular design incorporating specific chimeric protein–receptor interactions is an essential aspect of understanding biological mechanisms and precision modeling of molecular complexes. In this study, a detailed in-silico analysis was performed on a chimeric fusion protein designed to target Alzheimer’s disease. The fusion protein was created using the amino acid sequences retrieved from the beta-NGF, Nerve Growth Factor protein and the A[Formula: see text] peptide, with a rigid linker in place that ensures structural integrity. Its physicochemical characterization was predicted using ProtParam along with the evaluation of its secondary and tertiary structures, while its potential toxicity and allergenicity were analyzed through online analytical tools. The structural integrity of the fusion protein was evaluated through Rampage and ERRAT analyses, resulting in an ERRAT quality factor of 85 and indicating that 98.6% of the residues were placed within favored regions as suggested by the corresponding Ramachandran plot. Docking experiments were conducted using the HDOCK Server, followed by molecular simulations with the OpenMM engine, employing the AMBER force field for evaluation. The quality, validity, interaction analysis and stability of the fusion protein reveal that it indeed represents a functional molecular entity. The fact that the beta-NGF-A[Formula: see text] fusion gene is cloned and expressed in a suitable prokaryotic system may indicate that this is a very promising candidate for novel therapeutic approaches that target Alzheimer’s disease.

Impact of Dehydration Processing on Scallop (Patinopecten yessoensis) Adductor Muscle: Structural and Oxidative Insights

This study investigated the impact of four drying techniques—hot air drying (HAD), vacuum hot air drying (VFAD), microwave drying (MWD), and vacuum freeze-drying (VFD)—on the structural, physicochemical, and functional properties of scallop adductor muscles, a critical marine resource in the food industry. The results demonstrated that VFD optimally preserved the ultrastructural integrity of the tissue, maintaining its surface fibrous architecture and achieving a superior recovery ration (78%) and rehydration ration (186.5%) compared to HAD, VFAD, and MWD. While the zeta potential remained statistically invariant across methods, HAD induced the largest particle agglomeration, followed by MWD. Notably, VFD enhanced protein stability, increasing the sulfhydryl content by 163.2% and reducing carbonyl formation by 48.1% relative to HAD, whereas MWD had the opposite effect. Multispectral analyses revealed the severe disruption of protein secondary and tertiary structures after MWD, while VFD minimized conformational denaturation. Statistical modeling ranked the drying sensitivity parameters as follows: surface hydrophobicity &gt; hardness&gt; β-turn content &gt; dityrosine crosslinking &gt; transverse relaxation time T23. These findings underscore VFD as the optimal method for mitigating structural degradation and oxidative damage in scallop processing, providing actionable insights to enhance the technofunctional quality of shelf-stable scallop products.

Single-cell transcriptomics analysis reveals that the tumor-infiltrating B cells determine the indolent fate of papillary thyroid carcinoma

ObjectiveActive surveillance (AS) offers a viable alternative to surgical intervention for the management of indolent papillary thyroid carcinoma (PTC), helping to minimize the incidence of unnecessary treatment. However, the broader adoption of AS is hindered by the need for more reliable diagnostic markers. This study aimed to identify the differences between indolent and progressive PTC and find new targets for biomarker development and therapeutic strategies.MethodsWe used single-cell RNA sequencing (scRNA-seq) to analyze cellular differences in 10 early-stage PTC tumors. Findings were validated in an additional 25 tumors using cell co-culture, migration assays, immunofluorescence staining, flow cytometry, and analysis of data from The Cancer Genome Atlas (TCGA).ResultsTumor-infiltrating B cells (TIL-B), particularly germinal center B cells (GC-B), were more abundant in indolent PTC. These cells suppressed thyroid cell proliferation in both indolent and progressive cases, though indolent PTC had a higher capacity to recruit peripheral B cells. In indolent cases, TIL-B cells showed increased proliferation and formed clusters within tertiary lymphoid structures (TLS). PTPRC-CD22 interactions were identified as potential drivers of TIL-B cell proliferation. Markers linked to GC-B cells, such as LMO2, were highlighted as potential diagnostic and prognostic indicators for indolent PTC.ConclusionThis study provides insights into the cellular landscape of early-stage PTC, revealing distinct tumor and immune microenvironment features in indolent and progressive cases. These findings advance the understanding of indolent PTC biology and support the development of reliable diagnostic and prognostic biomarkers.

Prognostic implications and characterization of tumor-associated tertiary lymphoid structures genes in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is among the most aggressive cancers, with rising incidence and limited responsiveness to immunotherapy due to its highly suppressive tumor microenvironment (TME). Tertiary lymphoid structures (TLS), ectopic formation structures of immune cells, are linked to better prognosis and improved immunotherapy responses in PDAC. Understanding TLS’s role in PDAC could enhance immunotherapy effectiveness.MethodsThis study integrated transcriptomic and clinical data from 310 PDAC patients in GEO database. We performed consensus clustering using tumor-associated TLS (TA-TLS) genes, identifying three distinct molecular subtypes. Single-sample gene set enrichment analysis (ssGSEA) was then employed to calculate a TLS score for each patient, allowing for TLS-based evaluation. Key prognostic genes were identified using an iterative LASSO method, leading to the construction of a risk assessment model, which was validated across independent cohorts. We further analyzed the TLS score using single-cell RNA sequencing (scRNA-seq), visualized key gene expression, and validated protein expression through immunohistochemistry (IHC). Additionally, we explored the effects of DNASE1L3 on cell proliferation and migration, and its immune-related functions using Gene Set Enrichment Analysis (GSEA) and multiplex cytokine analysis.ResultsConsensus clustering revealed three PDAC molecular subtypes with significant differences in prognosis, TA-TLS gene expression, and TME features. The TLS score effectively stratified patients into high and low groups, correlating with survival outcomes and TME characteristics. Our risk model, validated across cohorts, reliably predicted patient outcomes. Validation studies showed lower expression of DNASE1L3 and IL33 in tumor tissues. scRNA-seq confirmed TLS score associations with immune cells. DNASE1L3 overexpression inhibited PDAC cell proliferation and migration, with cytokine analysis indicating increased immune activity.ConclusionsThis study elucidated the expression profile of TA-TLS genes in PDAC, constructed a TLS gene-based scoring system, and developed a related risk model. We also explored the functions and potential antitumor mechanisms of key genes, providing evidence and new insights for enhancing TLS-targeted immunotherapy strategies in PDAC.

Computational design of a fimbriae-derived multi-epitope vaccine candidate against Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen that causes infections in various parts of the body, with high mortality rates reported in antibiotic-resistant cases. Treating at-risk individuals requires crucial vaccination efforts due to the challenges that exist. This research involved designing a multi-epitope vaccine from K. pneumoniae's fimbriae antigens. Optimal T-cell and B-cell epitopes were chosen through in silico studies including epitope-HLAs molecular docking. The multi-epitope was created, featuring antigenic T- and B-cell epitopes, β-defensin as an adjuvant, the PADRE sequence to boost immunogenicity and well-suited linkers. The tertiary structure of the multi-epitope was achieved through modeling and molecular dynamics-based refinements. The construct underwent scrutiny for structural traits, physicochemical properties, conformational B epitope prediction, immune responses simulation, in silico cloning, molecular docking for assay binding to toll-like receptors (TLRs), and deformability studies. The outcomes indicated the vaccine candidate's positive attributes, encompassing immunogenicity, structure, physicochemical properties, solubility, TLR binding, toxicity, stability, allergenicity, and cross-reactivity. The multi-epitope vaccine candidate exhibits the potential for provoking diverse immune responses against K. pneumoniae. Nevertheless, additional invitro and invivo experimental tests are necessary to substantiate its efficacy.

The R337C mutation in the p53 oligomerization domain affects the regulatory domain and its ability to bind response elements: Evidence based on structural and biophysical studies.

The R337C mutation in the p53 oligomerization domain affects the regulatory domain and its ability to bind response elements: Evidence based on structural and biophysical studies.

Improving Aptamer Affinity and Determining Sequence-Activity Relationships via Motif-SELEX.

The affinity of nucleic acid aptamers isolated in vitro via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is often limited because the entire potential sequence space cannot be screened. In this study, we introduce Motif-SELEX, a novel method that enables the optimization of existing underperforming aptamers by generating libraries that broadly represent both the sequence and length variations of the parent sequence. This approach enables the isolation of sequences with improved affinity without the biases and limitations of traditional mutagenesis methods like doped SELEX and error-prone PCR. As a demonstration, we applied Motif-SELEX to a DNA-based morphine aptamer and a 2' fluoro- and methoxy-RNA-based apixaban aptamer, discovering new, better-performing sequences with differing random domain lengths from their parents and up to 10-fold improvements in affinity. These new sequences would be inaccessible to traditional post-SELEX methods. Critically, our analysis of Motif-SELEX pools also enabled us to identify sequence and structural elements crucial for target binding and to predict secondary and tertiary structures for a given aptamer family─even when those structures involve noncanonical nucleotide interactions. We believe that Motif-SELEX offers an effective and generalizable solution for optimizing the structure and binding properties of functional nucleic acid molecules for diverse applications.

Heme Regulatory Motif of Heme Oxygenase-2 Is Involved in the Interaction with NADPH-Cytochrome P450 Reductase and Regulates Enzymatic Activity.

Mammalian heme oxygenase (HO) catalyzes heme degradation using reducing equivalents supplied by NADPH-cytochrome P450 reductase (CPR). The tertiary structure of the catalytic domain of a constitutively expressed isoform of HO, HO-2, resembles that of the inductive isoform, HO-1, whereas HO-2 has two heme regulatory motifs (HRM) at the proximal portion of the C-terminus, where the disulfide linkage reflects cellular redox conditions and the second heme binding site is located. Here, we report the results of crosslinking experiments, which suggest that HRM is located near the FMN-binding domain of the CPR when it is complexed with HO-2. The enzymatic assay and reduction kinetics results suggest that heme-bound HRM negatively regulates HO-2 activity in vitro. Cellular redox conditions and free heme concentrations may regulate HO-2 activity.

Transfer learning radiomic model predicts intratumoral tertiary lymphoid structures in hepatocellular carcinoma: a multicenter study

BackgroundIntratumoral tertiary lymphoid structures (iTLS) in hepatocellular carcinoma (HCC) are associated with improved survival and may influence treatment decisions. However, their non-invasive detection remains challenging in HCC. We aim to...

Identification of potential plant secondary metabolites targeting begomovirus-associated betasatellite virulence factor βC1 protein through molecular docking, simulation and MM-PBSA studies

The βC1 protein encoded by begomoviruses plays a key role in counteracting plant defense mechanisms, making it a prime target for antiviral compounds. Here, we investigated the role of plant secondary metabolites for their antiviral potential. Initially, the tertiary structure of βC1 protein was predicted and docked to 2851 plant secondary metabolites along with positive controls ribavirin and ningnanmycin. Additionally, 43 analogues of the ligand with highest dock score were created and assessed against the βC1 protein to see if they offered better binding energy than the initial compound. To check the protein–ligand complex's stability, we performed a 100 ns molecular dynamics (MD) simulation, then measured the change in Gibbs free energy (ΔG) using the molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) approach. Our study found that Anonaine, a bioactive compound, had a high binding score of − 6.0 kcal/mol, with its analogues a33 and a41 scoring − 6.8 kcal/mol and − 6.6 kcal/mol, respectively. Molecular dynamics simulations for these complexes showed stable interactions, with minimal changes in root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values. Additionally, the protein–ligand complexes showed consistent hydrogen bond formation, with negligible changes in the radius of gyration (Rg) and solvent-accessible surface area (SASA). The binding free energy (ΔG) calculations revealed estimated values of − 16.99 ± 2.16, − 18.29 ± 1.91, and − 27.26 ± 2.11 for the βC1-Anonaine, βC1-a33, and βC1-a44 protein–ligand complexes, respectively. In silico toxicity predictions suggest top ligands are less toxic, making them ideal for biopesticide development. Overall, these findings reveal the potential of plant secondary metabolites as promising agents in controlling begomovirus infections.Graphical

Antigen-presenting cancer associated fibroblasts enhance antitumor immunity and predict immunotherapy response.

Cancer-associated fibroblasts (CAF) play a crucial role in tumor progression and immune regulation. However, the functional heterogeneity of CAFs remains unclear. Here, we identify antigen-presenting CAFs (apCAF), characterized by high MHC II expression, in gastric cancer (GC) tumors and find that apCAFs are preferentially located near tertiary lymphoid structures. Both in vivo and in vitro experiments demonstrate that apCAFs promote T cell activation and enhances its cytotoxic and proliferative capacities, thereby strengthening T cell-mediated anti-tumor immunity. Additionally, apCAFs facilitate the polarization of macrophages toward a pro-inflammatory phenotype. These polarized macrophages, in turn, promote the formation of apCAFs, creating a positive feedback loop that amplifies anti-tumor immune responses. Notably, baseline tumors in immunotherapy responders across various cancer types exhibit higher levels of apCAFs infiltration. This study advances the understanding of CAFs heterogeneity in GC and highlights apCAFs as a potential biomarker for predicting immunotherapy response in pan-cancer.

Spatial distribution of tertiary lymphoid structures in the molecular and clinical context of non-small cell lung cancer.

Tertiary lymphoid structures (TLS) are lymphocyte aggregates resembling secondary lymphoid organs and are pivotal in cancer immunity. The ambiguous morphological definition of TLS makes it challenging to ascertain their clinical impact on patient survival and response to immunotherapy. This study aimed to characterize TLS in hematoxylin-eosin tissue sections from lung cancer patients, assessing their occurrence in relation to the local immune environment, mutational background, and patient outcome. Two pathologists evaluated one whole tissue section from resection specimens of 680 NSCLC patients. TLS were spatially quantified within the tumor area or periphery and further categorized based on the presence of germinal centers (mature TLS). Metrics were integrated with immune cell counts, genomic and transcriptomic data, and correlated with clinical parameters. TLS were present in 86% of 536 evaluable cases, predominantly in the tumor periphery, with a median of eight TLS per case. Mature TLS were found in 24% of cases. TLS presence correlated positively with increased plasma cell (CD138+) and lymphocytic cell (CD3+, CD8+, FOXP3+) infiltration. Tumors with higher tumor mutational burden exhibited higher numbers of peripheral TLS. The overall TLS quantity was independently associated with improved patient survival, irrespective of TLS maturation status. This prognostic association held true for peripheral TLS but not for tumor TLS. TLS in NSCLC is common and their correlation with a specific immune phenotype suggests biological relevance in the local immune reaction. The prognostic significance of this scoring system on routine hematoxylin-eosin sections has the potential to augment diagnostic algorithms for NSCLC patients.

A Comparison of the Structural Changes and IgG Immunobinding Activity of Parvalbumin in Salangid Icefish (Neosalanx taihuensis) After Glycation and Ultra-High Pressure Treatment

The aim of this study was to compare the effects of glycation and ultra-high pressure (UHP) treatment on the structure and IgG immunobinding activity of Salangidae icefish PV. The Circular Dichroism (CD) and Fluorescence Spectroscopy (FS) findings indicated that the glycation significantly affected both the secondary and tertiary structures of PV. However, the impact of UHP processing on the structure of PV was found to be less significant compared to the glycation. Western Blot analysis also revealed that the glycation markedly reduced the antigen specificity of PV. Conversely, UHP treatments at 300 MPa and 400 MPa slightly decreased the antigen specificity, whereas lower or excessively high pressures did not have a substantial impact. This research contributes valuable insights into strategies for reducing the allergenic potential of Salangid icefish.

Multi-group structure analysis and molecular docking of aptamers and small molecules: A case study of chloramphenicol.

Multi-group structure analysis and molecular docking of aptamers and small molecules: A case study of chloramphenicol.

Tissue-specific expression, functional analysis, and polymorphism of the KRT2 gene in sheep horn.

Tissue-specific expression, functional analysis, and polymorphism of the KRT2 gene in sheep horn.

Proteome-wide assessment of differential missense variant clustering in neurodevelopmental disorders and cancer.

Proteome-wide assessment of differential missense variant clustering in neurodevelopmental disorders and cancer.

Two Nonsense GLI3 Variants Are Identified in Two Chinese Families With Polydactyly.

Polydactyly is a prevalent limb deformity with an autosomal dominant inheritance pattern, manifesting in both syndromic and nonsyndromic forms. It exhibits significant etiological and clinical diversity. This study aims to identify the pathogenic cause in two patients with sub-PHS (sub-Pallister-Hall Syndrome) and PAP (postaxial polydactyly), respectively, from two Chinese pedigrees. Exome sequencing was performed on patients to screen for potential pathogenic variants. Subsequently, these variants were validated by Sanger sequencing. The c.3342dupC and c.4431dupT mutant plasmids were transfected into HEK293T cells, and the effects of GLI3 mutations on transcription and protein levels were analyzed via qRT-PCR and Western blot. Additionally, Swiss Model was utilized to predict the effects of mutations on protein tertiary structure. The mutations of GLI3 (NM_000168.6: c.3342dupC; p. A1115Rfs*14) (NM_000168.6: c.4431dupT; p. Glu1478Ter) were identified in affected individuals. These mutations were present exclusively in the patients and absent in the healthy individuals. No significant difference in transcription levels between the mutations and wild type was observed. Functional analysis revealed that the truncated variants p. A1115Rfs*14 and p. Glu1478Ter exhibited reduced molecular weight and potential functional impairment due to protein retention. The mutations p. A1115Rfs*14 and p. Glu1478Ter in GLI3 may account for sub-PHS and PAP in the two patients, respectively. This finding expands the mutation and phenotype spectrum associated with GLI3, providing valuable insights for the clinical diagnosis of polysyndactyly.