Proteomes

25 pages, 1984 KiB

Open AccessReview

Advances in the Study of Protein Deamidation: Unveiling Its Influence on Aging, Disease Progression, Forensics and Therapeutic Efficacy

by Sunil S. Adav

Proteomes 2025, 13(2), 24; https://doi.org/10.3390/proteomes13020024 - 5 Jun 2025

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Abstract

Protein deamidation, a nonenzymatic post-translational modification that converts asparagine and glutamine residues into their acidic forms, such as aspartic acid, iso-aspartic acid, or glutamic acid, has emerged as a pivotal process affecting protein stability and function. Once considered a minor biochemical occurrence, deamidation [...] Read more.

Protein deamidation, a nonenzymatic post-translational modification that converts asparagine and glutamine residues into their acidic forms, such as aspartic acid, iso-aspartic acid, or glutamic acid, has emerged as a pivotal process affecting protein stability and function. Once considered a minor biochemical occurrence, deamidation is now recognized for its significant role in aging, age-associated diseases, disease progression, cancer, and therapeutic efficacy. This review explores the recent advances in understanding protein deamidation, its impact on cellular homeostasis, protein misfolding, and age-related and chronic diseases including neurodegeneration and cancer. The study also highlights the challenges posed by deamidation in biopharmaceuticals, where it compromises therapeutic stability and efficacy. Advancements in state-of-the-art analytical techniques and computational approaches for identifying deamidation sites and predicting deamidation-prone regions are discussed, along with deeper insights into how deamidation affects protein structure and function. Based on the current insights, this review underscores the dual role of deamidation as both a natural regulatory process and a contributor to pathological states, providing a roadmap for future research in aging biology, disease mechanisms, and therapeutics. Full article

(This article belongs to the Special Issue Proteomics in Chronic Diseases: Issues and Challenges)

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13 pages, 1026 KiB

Open AccessArticle

A Clinical Validation of a Diagnostic Test for Esophageal Adenocarcinoma Based on a Novel Serum Glycoprotein Biomarker Panel: PromarkerEso

by Jordana Sheahan, Iris Wang, Peter Galettis, David I. Watson, Virendra Joshi, Michelle M. Hill, Richard Lipscombe, Kirsten Peters and Scott Bringans

Proteomes 2025, 13(2), 23; https://doi.org/10.3390/proteomes13020023 - 4 Jun 2025

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Abstract

Background: Esophageal adenocarcinoma (EAC) diagnosis involves invasive and expensive endoscopy with biopsy, but rising EAC incidence has not been reduced by increased surveillance. This study aimed to develop and clinically validate a novel glycoprotein biomarker blood test for EAC, named PromarkerEso. Methods: Serum [...] Read more.

Background: Esophageal adenocarcinoma (EAC) diagnosis involves invasive and expensive endoscopy with biopsy, but rising EAC incidence has not been reduced by increased surveillance. This study aimed to develop and clinically validate a novel glycoprotein biomarker blood test for EAC, named PromarkerEso. Methods: Serum glycoprotein relative concentrations were measured using a lectin-based magnetic bead array pulldown method, with multiple reaction monitoring mass spectrometry in 259 samples across three independent cohorts. A panel of glycoproteins: alpha-1-antitrypsin, alpha-1-antichymotrypsin, complement C9 and plasma kallikrein, were combined with clinical factors (age, sex and BMI) in an algorithm to categorize the samples by the risk of EAC. Results: PromarkerEso demonstrated a strong discrimination of EAC from the controls (area under the curve (AUC) of 0.91 in the development cohort and 0.82 and 0.98 in the validation cohorts). The test exhibited a high sensitivity for EAC (98% in the development cohort, and 99.9% and 91% in the validation cohorts) and a high specificity (88% in the development cohort, and 86% and 99% in the validation cohorts). PromarkerEso identified individuals with and without EAC (96% and 95% positive and negative predictive values). Conclusions: This less invasive approach for EAC detection with the novel combination of these glycoprotein biomarkers and clinical factors coalesces in a potential step toward improved diagnosis. Full article

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14 pages, 3432 KiB

Open AccessArticle

Chromosome X Open Reading Frame 38 (CXorf38) Is a Tumor Suppressor and Potential Prognostic Biomarker in Lung Adenocarcinoma: The First Characterization

by Rui Yan, Heng-Wee Tan, Na-Li Cai, Le Yu, Yan Gao, Yan-Ming Xu and Andy T. Y. Lau

Proteomes 2025, 13(2), 22; https://doi.org/10.3390/proteomes13020022 - 3 Jun 2025

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Abstract

Background: Previously, we found that an uncharacterized protein CXorf38 is significantly downregulated in human ZIP8-knockout (KO) cells. Given that ZIP8 regulates essential micronutrients linked to diseases including cancer, this study aims to characterize CXorf38 and evaluate its role in lung adenocarcinoma. Methods: iTRAQ-based [...] Read more.

Background: Previously, we found that an uncharacterized protein CXorf38 is significantly downregulated in human ZIP8-knockout (KO) cells. Given that ZIP8 regulates essential micronutrients linked to diseases including cancer, this study aims to characterize CXorf38 and evaluate its role in lung adenocarcinoma. Methods: iTRAQ-based proteomics was previously used to identify the abundance of proteins in ZIP8-KO cells. Cell proliferation and colony formation assays were used to examine the function of CXorf38 by overexpressing the gene in lung adenocarcinoma cell lines. Kaplan–Meier survival analysis was used to assess the prognostic value of CXorf38, while TCGA clinical database analysis was used to evaluate its expression in lung cancer tissues, particularly in smokers. Bioinformatics analyses (GO, KEGG, PPI, and ICI) were performed on CXorf38-coexpressed genes derived from patients with lung cancer. Results: CXorf38 overexpression suppressed lung cancer cell proliferation and colony formation, suggesting a tumor-suppressive role. Higher CXorf38 expression correlated with improved survival in patients with lung adenocarcinoma, but not in lung squamous cell carcinoma. Clinical data showed CXorf38 downregulation with lung cancer tissues of smokers, indicating a potential role in smoking-induced cancer progression and treatment. Functional analysis using bioinformatics linked CXorf38 to immune response regulation, suggesting involvement in the tumor immune microenvironment. Conclusions: Our study reveals for the first time that CXorf38 is a potential tumor suppressor, prognostic biomarker, and/or tumor immune regulator in lung adenocarcinoma—further research is warranted to explore its role in tumor immunity and its therapeutic potential. Full article

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17 pages, 562 KiB

Open AccessReview

Oxidative Stress and Its Role in the Emergence and Progression of Myelodysplastic Syndromes: Insights from Proteomic Analysis and Other Methodologies

by Anastasia Boura-Theodorou, Konstantina Psatha, Stefania Maniatsi, Areti Kourti, Georgia Kaiafa, Michalis Aivaliotis and Kali Makedou

Proteomes 2025, 13(2), 21; https://doi.org/10.3390/proteomes13020021 - 3 Jun 2025

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Abstract

Myelodysplastic syndromes (MDS) belong to a category of malignant stem-cell and myeloid disorders that deteriorate the function of the hematopoietic system exacerbated by the omnipresent anemia that characterizes myelodysplasia. The pathogenesis of MDS is driven by cytogenetic abnormalities along with the excessive production [...] Read more.

Myelodysplastic syndromes (MDS) belong to a category of malignant stem-cell and myeloid disorders that deteriorate the function of the hematopoietic system exacerbated by the omnipresent anemia that characterizes myelodysplasia. The pathogenesis of MDS is driven by cytogenetic abnormalities along with the excessive production of pro-inflammatory cytokines and disruptions in inflammatory signaling pathway, particularly through the influence of carbonylated proteins, which are linked to MDS progression. An additional and major contributor to the pathogenesis of MDS is oxidative stress marked by uncontrolled levels of reactive oxygen species (ROS), which have been suggested as potential biomarkers for assessing disease severity and stratifying MDS cases throughout a variety of methods. Excessive and non-accumulative levels of free iron can also lead to iron overload (IOL)—related promotion of a high oxidative state, whether we refer to treatment-related IOL or natural IOL mechanisms. Proteomic analysis has emerged as a powerful tool for profiling protein samples, and, consequently, understanding the molecular changes underlying MDS. In this review, we evaluated studies and their methodologies aiming in investigating distinctive proteomics signatures associated with MDS pathogenesis, focusing on the role of oxidative stress at the protein level. Full article

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21 pages, 3415 KiB

Open AccessArticle

Knowledge Discovery in Databases of Proteomics by Systems Modeling in Translational Research on Pancreatic Cancer

by Mathilde Resell, Elisabeth Pimpisa Graarud, Hanne-Line Rabben, Animesh Sharma, Lars Hagen, Linh Hoang, Nan T. Skogaker, Anne Aarvik, Magnus K. Svensson, Manoj Amrutkar, Caroline S. Verbeke, Surinder K. Batra, Gunnar Qvigstad, Timothy C. Wang, Anil Rustgi, Duan Chen and Chun-Mei Zhao

Proteomes 2025, 13(2), 20; https://doi.org/10.3390/proteomes13020020 - 29 May 2025

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Abstract

Background: Knowledge discovery in databases (KDD) can contribute to translational research, also known as translational medicine, by bridging the gap between in vitro and in vivo studies, and clinical applications. Here, we propose a ‘systems modeling’ workflow for KDD. Methods: This framework includes [...] Read more.

Background: Knowledge discovery in databases (KDD) can contribute to translational research, also known as translational medicine, by bridging the gap between in vitro and in vivo studies, and clinical applications. Here, we propose a ‘systems modeling’ workflow for KDD. Methods: This framework includes the data collection of a composition model (various research models), processing model (proteomics) and analytical model (bioinformatics, artificial intelligence/machine leaning and pattern evaluation), knowledge presentation, and feedback loops for hypothesis generation and validation. We applied this workflow to study pancreatic ductal adenocarcinoma (PDAC). Results: We identified the common proteins between human PDAC and various research models in vitro (cells, spheroids and organoids) and in vivo (mouse mice). Accordingly, we hypothesized potential translational targets on hub proteins and the related signaling pathways, PDAC-specific proteins and signature pathways, and high topological proteins. Conclusions: This systems modeling workflow can be a valuable method for KDD, facilitating knowledge discovery in translational targets in general, and in particular to PADA in this case. Full article

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16 pages, 3788 KiB

Open AccessArticle

Unraveling the Central Role of Global Regulator PprI in Deinococcus radiodurans Through Label-Free Quantitative Proteomics

by Siyu Zhu, Feng Liu, Hao Wang and Yongqian Zhang

Proteomes 2025, 13(2), 19; https://doi.org/10.3390/proteomes13020019 - 23 May 2025

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Abstract

Background: Deinococcus radiodurans, renowned for its exceptional resistance to radiation, provides a robust model for elucidating cellular stress responses and DNA repair mechanisms. Previous studies have established PprI as a key regulator contributing to radiation resistance through its involvement in DNA damage [...] Read more.

Background: Deinococcus radiodurans, renowned for its exceptional resistance to radiation, provides a robust model for elucidating cellular stress responses and DNA repair mechanisms. Previous studies have established PprI as a key regulator contributing to radiation resistance through its involvement in DNA damage repair pathways, oxidative stress response, and metabolic regulation. Methods: Building upon these foundations, our study employs label-free quantitative (LFQ) proteomics coupled with high-resolution mass spectrometry to systematically map pprI deletion protein networks by comparing the global proteomic profiles of pprI knockout and wild-type D. radiodurans strains. Results: Under stringent screening criteria, we identified 719 significantly higher and 281 significantly lower abundant proteins in the knockout strain compared to wild-type strains. Functional analysis revealed that PprI deficiency disrupts homologous recombination (HR) repair, activates nucleotide excision repair (NER) and base excision repair (BER) as a compensatory mechanism, and impairs Mn/Fe homeostasis and carotenoid biosynthesis, leading to increased oxidative stress. Furthermore, PprI deficiency induces significant metabolic reprogramming, including impaired purine synthesis, compromised cell wall integrity, etc. Conclusions: These proteomic findings delineate the extensive regulatory network influenced by PprI, revealing coordinated perturbations across multiple stress response systems when PprI is absent. Full article

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20 pages, 1967 KiB

Open AccessArticle

Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling

by Anu Jain, Rafaela Muniz de Queiroz, Jayanta K. Chakrabarty, Karl A. T. Makepeace, Carol Prives and Lewis M. Brown

Proteomes 2025, 13(2), 18; https://doi.org/10.3390/proteomes13020018 - 22 May 2025

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Abstract

Background: We utilized data-independent acquisition (DIA) to study the poorly understood biology of Mdm2 and MdmX in a p53-null context. Mdm2 and MdmX form an E3-ligase complex that has as its most well-studied function the negative regulation of the tumor suppressor p53; however, [...] Read more.

Background: We utilized data-independent acquisition (DIA) to study the poorly understood biology of Mdm2 and MdmX in a p53-null context. Mdm2 and MdmX form an E3-ligase complex that has as its most well-studied function the negative regulation of the tumor suppressor p53; however, it is also known to interact with many other proteins in a p53-independent manner. Methods: In this work, small-molecule and siRNA-based technology were used to modify Mdm2/MdmX activity in a human non-small-cell lung carcinoma cell line lacking p53 expression. Study of the proteome of these cells helped identify biological processes where Mdm2 and MdmX may play roles in a p53-independent manner. Proteins from H1299 cells, treated with the drug MEL23 or siRNA against Mdm2 or MdmX, were analyzed. Results: Protein ontology and function were analyzed, revealing which pathways are affected by modulation of the proteins that form the complex. Insights into how those functions are dependent on the activity of the complex also gained via comparisons among the three groups of samples. Conclusions: We selected a potential target from the DIA analysis and validated it by immunoblotting and qPCR, and this allows us to demonstrate a new interaction partner of the Mdm2-MdmX complex in human cells. Full article

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24 pages, 12086 KiB

Open AccessArticle

Integrative Spatial Proteomics and Single-Cell RNA Sequencing Unveil Molecular Complexity in Rheumatoid Arthritis for Novel Therapeutic Targeting

by Xue Wang, Fei Wang, Archana S. Iyer, Heather Knight, Lori J. Duggan, Yingli Yang, Liang Jin, Baoliang Cui, Yupeng He, Jan Schejbal, Lucy A. Phillips, Bohdan P. Harvey, Sílvia Sisó and Yu Tian

Proteomes 2025, 13(2), 17; https://doi.org/10.3390/proteomes13020017 - 22 May 2025

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Abstract

Understanding the heterogeneity of Rheumatoid Arthritis (RA) and identifying therapeutic targets remain challenging using traditional bulk transcriptomics alone, as it lacks the spatial and protein-level resolution needed to fully capture disease and tissue complexities. In this study, we applied Laser Capture Microdissection (LCM) [...] Read more.

Understanding the heterogeneity of Rheumatoid Arthritis (RA) and identifying therapeutic targets remain challenging using traditional bulk transcriptomics alone, as it lacks the spatial and protein-level resolution needed to fully capture disease and tissue complexities. In this study, we applied Laser Capture Microdissection (LCM) coupled with mass spectrometry-based proteomics to analyze histopathological niches of the RA synovium, enabling the identification of protein expression profiles of the diseased synovial lining and sublining microenvironments compared to their healthy counterparts. In this respect, key pathogenetic RA proteins like membrane proteins (TYROBP, AOC3, SLC16A3, TCIRG1, and NCEH1), and extracellular matrix (ECM) proteins (PLOD2, OGN, and LUM) showed different expression patterns in diseased synovium compartments. To enhance our understanding of cellular dynamics within the dissected regions, we further integrated the proteomic dataset with single-cell RNA sequencing (scRNA-seq), and deduced cell type enrichment, including T cells, fibroblasts, NK cells, myeloid cells, B cells, and synovial endothelial cells. By combining high-resolution spatial proteomics and transcriptomic analyses, we provide novel insights into the molecular mechanisms driving RA, and highlight potential protein targets for therapeutic intervention. This integrative approach offers a more comprehensive view of RA synovial pathology, and mitigates the limitations of traditional bulk transcriptomics in target discovery. Full article

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42 pages, 12845 KiB

Open AccessArticle

Intrinsic Disorder and Phase Separation Coordinate Exocytosis, Motility, and Chromatin Remodeling in the Human Acrosomal Proteome

by Shivam Shukla, Sean S. Lastorka and Vladimir N. Uversky

Proteomes 2025, 13(2), 16; https://doi.org/10.3390/proteomes13020016 - 28 Apr 2025

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Abstract

Intrinsic disorder refers to protein regions that lack a fixed three−dimensional structure under physiological conditions, enabling conformational plasticity. This flexibility allows for diverse functions, including transient interactions, signaling, and phase separation via disorder-to-order transitions upon binding. Our study focused on investigating the role [...] Read more.

Intrinsic disorder refers to protein regions that lack a fixed three−dimensional structure under physiological conditions, enabling conformational plasticity. This flexibility allows for diverse functions, including transient interactions, signaling, and phase separation via disorder-to-order transitions upon binding. Our study focused on investigating the role of intrinsic disorder and liquid−liquid phase separation (LLPS) in the human acrosome, a sperm-specific organelle essential for fertilization. Using computational prediction models, network analysis, Structural Classification of Proteins (SCOP) functional assessments, and Gene Ontology, we analyzed 250 proteins within the acrosomal proteome. Our bioinformatic analysis yielded 97 proteins with high levels (>30%) of structural disorder. Further analysis of functional enrichment identified associations between disordered regions overlapping with SCOP domains and critical acrosomal processes, including vesicle trafficking, membrane fusion, and enzymatic activation. Examples of disordered SCOP domains include the PLC-like phosphodiesterase domain, the t-SNARE domain, and the P-domain of calnexin/calreticulin. Protein–protein interaction networks revealed acrosomal proteins as hubs in tightly interconnected systems, emphasizing their functional importance. LLPS propensity modeling determined that over 30% of these proteins are high-probability LLPS drivers (>60%), underscoring their role in dynamic compartmentalization. Proteins such as myristoylated alanine-rich C-kinase substrate and nuclear transition protein 2 exhibited both high LLPS propensities and high levels of structural disorder. A significant relationship (p < 0.0001, R² = 0.649) was observed between the level of intrinsic disorder and LLPS propensity, showing the role of disorder in facilitating phase separation. Overall, these findings provide insights into how intrinsic disorder and LLPS contribute to the structural adaptability and functional precision required for fertilization, with implications for understanding disorders associated with the human acrosome reaction. Full article

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22 pages, 6198 KiB

Open AccessArticle

Small Extracellular Vesicle (sEV) Uptake from Lung Adenocarcinoma and Squamous Cell Carcinoma Alters T-Cell Cytokine Expression and Modulates Protein Profiles in sEV Biogenesis

by Hafiza Padinharayil, Jinsu Varghese, Pulikkottil Raphael Varghese, Cornelia M. Wilson and Alex George

Proteomes 2025, 13(2), 15; https://doi.org/10.3390/proteomes13020015 - 23 Apr 2025

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Abstract

Background: Despite advances in immunotherapy, non-small-cell lung carcinoma (NSCLC)’s clinical success is limited, possibly due to substantial immunological alterations in advanced cancer patients. This study examines the immunomodulatory effects of sEVs derived from lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) on T [...] Read more.

Background: Despite advances in immunotherapy, non-small-cell lung carcinoma (NSCLC)’s clinical success is limited, possibly due to substantial immunological alterations in advanced cancer patients. This study examines the immunomodulatory effects of sEVs derived from lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) on T cells. Methods: SEVs were isolated from lung cancer cell lines and Jurkat-E6.1. SEV size and morphology were analyzed by NTA and TEM, respectively, while Western blotting confirmed sEV markers. SEV uptake was assessed, followed by resazurin assay, RNA isolation, quantification, cDNA preparation, RT-PCR, nano LC-MS, and bioinformatic analysis, before and after treating Jurkat-E6.1 cells with sEVs from A549 and SKMES1. Results: Cancer-derived sEVs were efficiently internalized by immune cells, reducing T-cell viability. The real-time PCR analysis showed downregulation of KI67, BCL2, BAX, TNFA, IL6, TGFβ, and IL10, suggesting reduced proliferation, dysregulated apoptosis, and impaired inflammatory and immunosuppressive signaling, and the upregulation of GZMB and IL2 suggests retained cytotoxic potential but possibly dysfunctional T-cell activation. Proteomic analysis revealed 39 differentially abundant proteins (DAPs) in ADC-treated T cells and 276 in SCC-treated T cells, with 19 shared DAPs. Gene Ontology (GO) analysis of these DAPs highlighted processes such as sEV biogenesis, metabolic pathways, and regulatory functions, with ADC sEVs influencing NAD metabolism, ECM binding, and oxidoreductase activity, while SCC sEVs affected mRNA stability, amino acid metabolism, and cadherin binding. The cytoplasmic colocalization suggests the presence of these proteins in the cellular and extracellular lumen, indicating the potential of further release of these proteins in the vesicles by T cells. Conclusion: Lung cancer-derived sEVs regulate T-cell activities through immunoregulatory signaling. The molecular interactions between sEVs and immune cells can reveal novel tumor immune regulatory mechanisms and therapeutic targets. Full article

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13 pages, 2233 KiB

Open AccessArticle

Role of LIN28B in the Regulation of Ribosomal Biogenesis and Lipid Metabolism in Medulloblastoma Brain Cancer Cells

by Ahmed Maklad, Mohammed Sedeeq, Kaveh Baghaei, Richard Wilson, John A. Heath, Nuri Gueven and Iman Azimi

Proteomes 2025, 13(2), 14; https://doi.org/10.3390/proteomes13020014 - 27 Mar 2025

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Abstract

Background: Medulloblastoma (MB) is the most aggressive paediatric brain cancer, highlighting the urgent need for new diagnostic and prognostic biomarkers and improved treatments to enhance patient outcomes. Our previous study identified LIN28B, an RNA-binding protein, as a potential diagnostic and prognostic marker for [...] Read more.

Background: Medulloblastoma (MB) is the most aggressive paediatric brain cancer, highlighting the urgent need for new diagnostic and prognostic biomarkers and improved treatments to enhance patient outcomes. Our previous study identified LIN28B, an RNA-binding protein, as a potential diagnostic and prognostic marker for MB and a pharmacological target to inhibit MB cell proliferation and stemness. However, the specific role of LIN28B and its mechanism of action in MB had not been studied. Methods: This study assessed LIN28B’s role in Daoy MB cells using siRNA-mediated silencing. LIN28B silencing was achieved with Dharmacon ON-TARGETplus SMARTpool and confirmed by Western blotting. Proliferation and protein assays evaluated the cell metabolic activity and viability. A proteomics analysis was conducted to examine the effect of LIN28B knockdown on the MB cell protein expression profile. The intracellular lipid droplets were assessed using the Nile Red Staining Kit, and nucleolar B23 protein levels were assessed by immunofluorescence. Both were visualised with a high-content IN Cell Analyser 2200. Results: Effective LIN28B silencing (>80%) was achieved in each experiment. LIN28B knockdown reduced the MB cell viability, impaired ribosome biogenesis, and promoted cellular lipid accumulation, as supported by proteomics and cell-based assays. Conclusions: This study highlights LIN28B as a promising target for regulating MB cell growth, ribosomal biogenesis, and lipid metabolism. Full article

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25 pages, 3652 KiB

Open AccessArticle

Cell-Type-Specific Heat-Induced Changes in the Proteomes of Pollen Mother Cells and Microspores Provide New Insights into Tomato Pollen Production Under Elevated Temperature

by Priya Thapa, Jun Guo, Kajol Pradhan, Dibya Thapa, Sudhakar Madhavarapu, Jing Zou, Jesse Potts, Hui Li, Joshua O’Hair, Chen Wang, Suping Zhou, Yong Yang, Tara Fish and Theodore W. Thannhauser

Proteomes 2025, 13(2), 13; https://doi.org/10.3390/proteomes13020013 - 25 Mar 2025

Cited by 1 | Viewed by 588

Abstract

Background: Tomatoes are self-pollinating plants, and successful fruit set depends on the production of functional pollen within the same flower. Our previous studies have shown that the ‘Black Vernissage’ tomato variety exhibits greater resilience to heat stress in terms of pollen productivity compared [...] Read more.

Background: Tomatoes are self-pollinating plants, and successful fruit set depends on the production of functional pollen within the same flower. Our previous studies have shown that the ‘Black Vernissage’ tomato variety exhibits greater resilience to heat stress in terms of pollen productivity compared to the ‘Micro-Tom’ variety. Pollen productivity is determined by meiotic activity during microsporogenesis and the development of free microspores during gametogenesis. This study focused on identifying heat stress (HS)-induced proteomes in pollen mother cells (PMCs) and microspores. Methods: Tomato plants were grown under two temperature conditions: 26 °C (non-heat-treated control) and 37 °C (heat-treated). Homogeneous cell samples of meiotic PMCs (prior to the tetrad stage) and free microspores were collected using laser capture microdissection (LCM). The heat-induced proteomes were identified using tandem mass tag (TMT)–quantitative proteomics analysis. Results: The enrichment of the meiotic cell cycle in PMCs and the pre-mitotic process in free microspores confirmed the correlation between proteome expression and developmental stage. Under HS, PMCs in both tomato varieties were enriched with heat shock proteins (HSPs). However, the ‘Black Vernissage’ variety exhibited a greater diversity of HSP species and a higher level of enrichment compared to the ‘Micro-Tom’ variety. Additionally, several proteins involved in gene expression and protein translation were downregulated in PMCs and microspores of both varieties. In the PMC proteomes, the relative abundance of proteins showed no significant differences between the two varieties under normal conditions, with very few exceptions. However, HS induced significant differential expression both within and between the varieties. More importantly, these heat-induced differentially abundant proteins (DAPs) in PMCs are directly involved in meiotic cell division, including the meiosis-specific protein ASY3 (Solyc01g079080), the cell division protein kinase 2 (Solyc11g070140), COP9 signalosome complex subunit 1 (Solyc01g091650), the kinetochore protein ndc80 (Solyc01g104570), MORC family CW-type zinc finger 3 (Solyc02g084700), and several HSPs that function in protecting the fidelity of the meiotic processes, including the DNAJ chaperone (Solyc04g009770, Solyc05g055160), chaperone protein htpG (Solyc04g081570), and class I and class II HSPs. In the microspores, most of the HS-induced DAPs were consistently observed across both varieties, with only a few proteins showing significant differences between them under heat stress. These HS-induced DAPs include proteases, antioxidant proteins, and proteins related to cell wall remodeling and the generation of pollen exine. Conclusions: HS induced more dynamic proteomic changes in meiotic PMCs compared to microspores, and the inter-varietal differences in the PMC proteomes align with the effects of HS on pollen productivity observed in the two varieties. This research highlights the importance of the cell-type-specific proteomics approach in identifying the molecular mechanisms that are critical for the pollen developmental process under elevated temperature conditions. Full article

(This article belongs to the Section Plant Proteomics)

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Proteomes, Volume 13, Issue 2 (June 2025) – 12 articles

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