Biology subjects

Burton, J. B.

Publications and source records attributed to Burton, J. B..

11 recordsLinked to original sources

bioRxiv2026

Shifts in protein aggregate stability define proteostasis decline in the aging human brain

Loss of proteostasis and the accumulation of insoluble protein aggregates are features of aging across model organisms and occur in all major age-related neurodegenerative diseases; yet how aggregation proceeds during normal human brain aging remains unknown. Using detergent-fractionation proteomics, we show that human brain aging does not involve uniform aggregate accumulation; rather, the insoluble proteome undergoes asymmetric remodeling beginning in midlife. Maximum-stability aggregates decline sharply in old age whereas intermediate-stability aggregates accumulate gradually before accelerating after age 80. Intermediate-stability aggregates are prone to liquid-liquid phase separation and are enriched with Alzheimers disease plaque and tangle constituents. Proteasome and cytosolic chaperone abundance predict individual differences in aggregate burden as strongly as age, offering supportive evidence, in humans, for therapies targeting these pathways. These findings establish aggregate remodeling as a feature of normal brain aging and position the accumulation of intermediate-stability aggregates as a molecular event on the path to neurodegenerative disease.

neuroscience↗

bioRxiv2025

Investigation of Neurons and Microglia from APOE3 Induced Pluripotent Stem Cells using Data-Independent Acquisitions on a ZenoTOF 7600

Induced pluripotent stem cell (iPSC)-derived neurons and microglia are valuable human models for studying neurodegenerative diseases. Specifically, the apolipoprotein E4 (APOE4) gene is a major genetic risk factor for late-onset Alzheimers disease. Apolipoprotein E (APOE) alleles E2, E3 and E4 can be beneficial, neutral, or increase the risk of Alzheimers disease (AD). Here, we developed a proteomic workflow using data-independent acquisitions to provide a quantitative mass spectrometric proteome analysis, and proteomic screening assays for brain-specific cell types derived from iPSC. Protein groups were quantified in APOE3 neurons and microglia, respectively, with [~]80% overlap. Cell type-specific markers and enriched pathways reflected the specialized functions of each cell type, such as synaptic signaling in neurons and immune and inflammatory responses in microglia. The neuron-specific markers included proteins APP, CALB1, CALB2, DLGs, GAP43, NEFL, MAPs; while microglial markers included proteins AIF1, CDs, MMP9, and ITGAM. Ultimately, the combination of robust iPSC differentiation and sensitive proteomic screening assays described here provides a valuable platform for probing the cellular mechanisms underlying neurological disorders. SignificanceThe quantification of dysregulated proteins and pathways in patient-derived neurons and microglia can provide insights into disease etiology and progression. More broadly, this DIA approach enables deep proteome profiling of unique iPSC-derived cell models, increasing their utility for investigating disease biology and therapeutic development. We focused on iPSC models from two important cell types of the brain, excitatory neurons and microglia. We integrated the proteomes of these two cell types. These tools provide robust biological and mass spectrometric screening tools for future therapeutic interventions using disease-relevant human brain cell types or brain organoid models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/686128v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d7dorg.highwire.dtl.DTLVardef@17594d3org.highwire.dtl.DTLVardef@7dd894org.highwire.dtl.DTLVardef@1606db_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPresentation of a proteomic workflow using data-independent acquisitions to monitor and screen proteomes of iPSC-derived brain cell types. C_LIO_LIQuick MS Assays to determine protein profiles of iPSC-derived neurons and microglia. C_LIO_LICharacterization of different cell type proteomes from APOE3 iPSCs. C_LIO_LIRevealing of neuron-specific markers and microglia-specific markers by mass spectrometry. C_LIO_LIStep-by-step instructions for the set-up of the DIA-MS assays C_LI

neuroscience↗

bioRxiv2025

A Fibroblast State Choreographs an Epithelial YAP-dependent Regenerative Program Essential to (Pre)malignancy via ECM-mediated Mechanotransduction

Chronic lung injury generates metaplasia which occasionally, but ominously, progresses to squamous dysplasia and squamous lung cancer. To identify mechanisms through which disrupted tissue homeostasis contributes to malignant initiation and progression, we used in vivo and in vitro heterotypic recombinant models of human bronchial epithelial cells (hBECs) and fibroblasts. We demonstrate that injury-associated TGF-{beta} signaling creates a fibroblast state dependent upon HSP47 upregulation. These fibroblasts accumulated collagen, thus elevating tissue stiffness and activating mechanosignaling that sustained YAP-dependent embryonic-like, pro-malignant activities in adjacent hBECs. This Stress/Tension-Instructive Fibroblast (STIF) state, exhibited by stressed fibroblasts in premalignant and malignant lesions across multiple cancer types, was sufficient to reprogram disease-free hBECs to metaplasia and to drive hBECs with compromised tumor suppressor function to dysplasia, yet could be inhibited and reversed. STIFs suffice to activate epithelial phenotypes reminiscent of oncogene-mediated cell transformation and induce (pre)malignancy via increased force transmission, providing novel targets for prevention. Statement of significanceTissue injury creates a regenerative pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state which is sufficient to activate a YAP-dependent, pre-malignant program to induce or unmask pre-cancerous phenotypes in epithelial cells through mechanotransduction. Inhibition of STIF activity or mechanosignaling prevents metaplasia and progression to dysplasia. HighlightsO_LITissue injury creates a pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state in multiple organs that precedes and persists through cancer C_LIO_LISTIF signaling alone, working through fibroblasts and not epithelial cells, is sufficient to activate embryonic-like plasticity and induce epithelial pre-cancerous metaplastic lesions C_LIO_LISTIFs program (pre)malignant phenotypes in adjacent epithelial cells through mechanosignaling by activating YAP prior to tumor formation C_LIO_LIInhibiting STIFs or mechanosignaling prevents/reverts metaplasia and prevents progression to dysplasia C_LI

cancer biology↗

bioRxiv2025

Age-Dependent Increase and Dysregulation of the Splicing Machinery in Acute Myeloid Leukemia of Older Patients

Acute myeloid leukemia (AML) is characterized by age-related resistance to therapy and poor outcomes. Initial remission rates vary from 30-80% with current therapies, but relapses are common. The 5-year survival is 30% overall but less than 10% for those 60 years of age or older. Resistance to current therapies is the central clinical challenge. This study investigated how age influences the AML proteome in patients with increasing age. We analyzed bone marrow or leukapheresis samples from 14 AML patients, including 9 older ([≥] 66 years) and 5 younger ([≤] 58 years) individuals, using proteomic technologies (Data-Independent Acquisitions, DIA-MS). We identified 4,471 protein groups, of which 889 exhibited significant changes and regulation between the two age groups. Proteins upregulated in older patients were predominantly associated with DNA transcription and translation, while downregulated proteins were linked to metabolic, catabolic, and extracellular matrix or structural processes. Significant changes in older samples affected the splicing machinery. Significant alterations in several of these factors, suggests either dysregulated or elevated splicing activity and reduced transcription initiation factor activity in older AML patients. Our study provides novel insights into molecular changes during AML arising in the older adult, highlighting the importance of age-specific therapeutic strategies. Statement of Significance of the StudyThis study is significant as it investigates a devastating and highly aggressive cancer, acute myeloid leukemia (AML), which often presents in patients with very low 5-year survival rates (32%), however which is even lower in patients over 60 years old (10%). This study really focuses on the complications and severe outcome in older patients which is not well understood. This is particularly relevant as older AML patients often do not respond to the available treatments. Understanding the pathways and protein profiles that are dysregulated in older AML patients compared to younger AML patients may in the future help to develop more customized therapeutic interventions or allow for some precision treatments. AML patients were stratified into cohort groups by age (older vs younger) and the proteomic profiling highlighted the particularly strong dysregulation of the splicing machinery in the older AML patients. While alternative splicing is often observed in cancers, here the emphasis is pointed to the fact that the dysregulation of the spliceosome and other pathways are greatly exacerbated in the older AML patients, likely leading to even worse outcomes and poor response to treatments in the older patients.

cancer biology↗

bioRxiv2025

Proteomic Analysis of Breast Cancer Subtypes Identifies Stromal Contributions that Dictate Aggressive Malignant Behavior

Breast cancer manifests as multiple subtypes with distinct patient outcomes and treatment strategies. Here, we optimized proteomic analysis of Formalin-Fixed Paraffin-Embedded (FFPE) specimens from patients diagnosed with five breast cancer subtypes, luminal A, luminal B, Her2, triple negative (TNBC) and metaplastic breast cancers (MBC), and from disease-free individuals undergoing reduction mammoplasty (RM). We identified and quantified [~]6,000 protein groups (with >2 peptides per protein) with significant changes in over 26% of proteins comparing each cancer subtype with control RM. Stringent statistical filters allowed us to deeply mine 576 significant conserved protein changes shared by all subtypes and protein changes unique to each subtype. The most aggressive subtype, MBC, revealed exacerbated stromal stress responses, as illustrated by a collagenolytic extracellular matrix (ECM) and immune participation biased towards neutrophils and eosinophils. Immunostaining of breast tissue sections confirmed differences across subtypes, in particular, a strong upregulation of SERPINH1, neutrophil-specific myeloperoxidase and eosinophil cationic protein in MBC. In summary, we present deep proteomic, digitalized protein abundance profiles, generated from FFPE breast cancer tissues, that revealed significant changes in ECM and cellular proteins. Statement of Significance of the StudyThis study is significant as it discovered deep proteomic signatures for the highly aggressive and malignant metaplastic breast cancer (MCB) which is now considered a fifth subtype based upon its remarkable intra-tumoral heterogeneity that illustrates its unique cell plasticity. To efficiently analyze formalin-fixed paraffin-embedded (FFPE) breast tissues from patients with different breast cancer subtypes and disease-free individuals, we optimized a novel workflow in which we combined paraffinization and Folch extraction. We identified and confidently quantified [~]6,000 protein groups. We were able to find robust changes in extracellular matrix (ECM) with cancer, even though no ECM enrichments were performed. Interestingly, despite the relatively small human cohort size (42 patients), distinct protein signatures emerged throughout all cancer subtypes - common and unique - with remarkable statistical significance for many cancer-relevant proteins and pathways. This Pilot study indicates the hypothesis that an altered stroma can dictate epithelial tumor cell fate. We also observed that MBC was characterized by an especially immunosuppressed tumor environment. We do note the limitation of the relatively small cohort size of our study, and in the future additional patient cohorts will be needed to further validate our findings.

cancer biology↗

bioRxiv2024

Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-{beta}-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21+ cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

cell biology↗

bioRxiv2024

Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures

Senescence emerged as significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. We present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified [~]1,300 exosome proteins released by senescent cells induced by three different senescence inducers. In parallel, a human plasma cohort from young (20-26 years) and old (65-74 years) individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. 247 lipids were identified in exosome samples. Following senescence induction, identified phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins increased significantly indicating cellular membrane changes. Interestingly, significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering therapeutic targets for interventions.

cell biology↗

bioRxiv2023

Dicarboxylic acid supplementation protects from acute kidney injury via stimulation of renal peroxisomal activity

IntroductionLysine succinylation is a post-translational modification associated with the control of several diseases, including acute kidney injury (AKI). It is suggested that hypersuccinylation favors peroxisomal fatty acid oxidation (FAO) instead of mitochondrial. In addition, the medium-chain fatty acids (MCFAs) dodecanedioic acid (DC12) and octanedioic acid (DC8), upon FAO, generate succinyl-CoA, resulting in hypersuccinylation. DC8 is convenient, inexpensive, easily administered, and efficient. We believe this study could be translated in the future to clinical settings, which would highly benefit patients at high risk of AKI. Methods and ResultsTo test the protective roles of MCFAs during AKI, mice were fed with control, 10% DC12, or 10% DC8 diet, then, subjected to either ischemic-AKI, or cisplatin-AKI models. Supplementation was provided until sacrifice. Biochemical, histologic, genetic, and proteomic analysis were performed, the latter involving a lysine-succinylome-based analysis. Both DC8 and DC12 prevented the rise of AKI markers in mice that underwent renal injury. However, DC8 was even more protective against AKI than DC12. Finally, succinylome analysis evidenced that the kidneys of DC8-fed mice showed an extensive succinylation of peroxisomal activity-related proteins, and a decline in mitochondrial FAO, in comparison to control-fed mice. ConclusionDC8 supplementation drives renal protein hypersuccinylation, promoting a shift from mitochondrial to peroxisomal FAO, and protecting against AKI. Significance StatementLysine succinylation of proteins is shown to control several diseases, including acute kidney injury (AKI). Here we show that mice supplemented with the medium-chain fatty acid octanedioic acid successfully presented a high level of succinylation and were protected from both ischemia-reperfusion- and cisplatin-induced AKI. Moreover, our study demonstrates that peroxisomal activity was increased while mitochondrial activity was preserved, suggesting that the metabolism of diet-obtained medium-chain fatty acids by peroxisomes is renoprotective.

pathology↗

bioRxiv2023

Substantial Downregulation of Mitochondrial and Peroxisomal Proteins during Acute Kidney Injury revealed by Data-Independent Acquisition Proteomics

Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A fast-acquisition rate ZenoTOF 7600 mass spectrometer was introduced for data-independent acquisition (DIA) for comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3,945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured mice exhibited severely declined health. The comprehensive and sensitive kidney-specific DIA assays highlighted here feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome and will serve as useful tools for developing novel therapeutics to remediate kidney function.

molecular biology↗

bioRxiv2022

Robust and Highly Efficient Extractions of Proteins from Bones enable Deep, High-Throughput Proteomic Quantification to Gain Insights into Bone Biology

Dysregulation of cell signaling in chondrocytes and in bone cells, such as osteocytes, osteoblasts, osteoclasts, and an elevated burden of senescent cells in cartilage and bone, are implicated in osteoarthritis (OA). Mass spectrometric analyses provides a crucial molecular tool-kit to understand complex signaling relationships in age-related diseases, such as OA. Here we introduce a novel mass spectrometric workflow to promote proteomic studies of bone and cartilage. This workflow uses highly specialized steps, including extensive overnight demineralization, pulverization, and incubation for 72 h in 6 M guanidine hydrochloride and EDTA, followed by proteolytic digestion. Analysis on a high-resolution Orbitrap Eclipse and Orbitrap Exploris 480 mass spectrometer using Data-Independent Acquisition (DIA) provides deep coverage of the bone proteome, and preserves post-translational modifications, such as hydroxyproline. A spectral library-free quantification strategy, directDIA, identified and quantified over 2,000 protein groups (with [≥] 2 unique peptides) from calcium-rich bone matrices. Key components identified were proteins of the extracellular matrix (ECM), bone-specific proteins (e.g., secreted protein acidic and cysteine rich, SPARC, and bone sialoprotein 2, IBSP), and signaling proteins (e.g., transforming growth factor beta-2, TGFB2), and lysyl oxidase homolog 2 (LOXL2), an important protein in collagen crosslinking. Post-translational modifications (PTMs) were identified without the need for specific enrichment. This includes collagen hydroxyproline modifications, chemical modifications for collagen self-assembly and network formation. Multiple senescence factors were identified, such as complement component 3 (C3) protein of the complement system and many matrix metalloproteinases, that might be monitored during age-related bone disease progression. Our innovative workflow yields in-depth protein coverage and quantification strategies to discover underlying biological mechanisms of bone aging and to provide tools to monitor therapeutic interventions. These novel tools to monitor the bone proteome open novel horizons to investigate bone-specific diseases, many of which are age-related.

biochemistry↗

bioRxiv2022

In-depth Analysis of the Sirtuin 5-regulated Mouse Brain Acylome using Library-free Data-Independent Acquisitions

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2,211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 171 malonylated and 640 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome. Statement of significance of the studyPost-translational modifications (PTMs) are key regulators of protein structure, functions, and interactions. A great variety of PTMs have been discovered, including lysine acylation, such as acetylation, malonylation, and succinylation. Lysine acylation is understudied, particularly in the brain, and analysis by mass spectrometry-based proteomics faces significant challenges. In this study, we present a robust and efficient workflow to investigate proteome-wide PTM remodeling combining affinity PTM enrichment and a novel spectral library-free data-independent acquisition (DIA) approach. The strength of label-free DIA becomes evident with the collection of comprehensive information by tandem mass spectrometry for all detectable precursor ions of all biological samples, and the highly accurate quantitative information that can subsequently be retrieved with time-efficient and straightforward library-free strategies. More importantly, this enables confident identification of PTM sites and differentiation of PTM isomers. We applied this workflow to decipher the malonylome and succinylome remodeling and cross-talk in brains from wild-type and Sirt5(-/-) mice, taking advantage of the demalonylase and desuccinylase activities of SIRT5, a nicotinamide adenine dinucleotide (NAD+)-dependent sirtuin. Interestingly, 10 malonylated proteins and 33 succinylated proteins targeted by SIRT5 are involved in the Parkinsons disease pathway, including subunit beta of the calcium/calmodulin-dependent protein kinase type II (Camk2b) and protein DJ-1 (Park7).

cell biology↗