Regulation Of Proteome Research Articles

Sex-biased proteomic regulation in obesity

Abstract Background Obesity is a major risk factor for heart failure with preserved ejection fraction, particularly in women. Obesity-associated inflammation is thought to contribute to this relationship. However, the role of biological sex in the regulation of circulating inflammatory proteins in obesity is not well understood. Purpose We aimed to explore sex differences in the plasma inflammatory protein profile in obese subjects. Methods Clinical data and samples were collected from 450 women and men with a body mass index (BMI) >27 kg/m² without known cardiovascular disease participating in the FAT associated CardiOvasculaR dysfunction study. Obesity was defined as BMI≥30 kg/m². Olink’s Target 96 inflammation panel, which profiles 92 proteins using proximity extension assay technology, was analysed in biobank samples. A comprehensive correlation analysis was performed to describe relationships between protein biomarkers and clinical variables. Gene enrichment analysis was performed to shed light on biological pathways associated with differentially expressed proteins (DEPs). To determine the functional significance of the DEPs, we used the repositories Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Pearson correlation coefficients were calculated to assess linear associations between the DEPs and relevant clinical variables. Results Women and men did not differ by age (49±9 vs 48±9 years), BMI (32.3±4.6 vs 31.6±3.7 kg/m²) or in prevalences of diabetes (both 11%) or obesity (65% vs 62%) (all p>0.05). However, hypertension was more common in men than women (74% vs 52%, p<0.001). In men (n=188), obesity was associated with downregulation of monocyte chemotactic protein 3, tumor necrosis factor (ligand) superfamily member 12, interleukin 10 and protein S100-A12 and with upregulation of neurotrophin-3 (all p<0.05), thereby suggesting a mechanism for the observed impaired inflammatory response and insulin resistance. In women (n=262), obesity was associated with the downregulation of interleukin 7, interleukin 12 subunit beta, interleukin 6, and C-X-C motif chemokine 11, suggesting a state of persistent low-grade inflammation. In addition, the upregulated sulfotransferase 1A1 and SIR2-like protein 2 proteins indicate a physiologic adaptation to oxidative stress and the metabolic excess endemic to obesity. Conclusion We found that the abundance of several circulating inflammatory proteins was altered in presence of obesity, and that the plasma proteomic profile differed by sex. Interestingly, there was no overlap in the DEPs between women and men in the presence of obesity. These findings support the hypothesis that the pathophysiological mechanisms driving obesity-associated inflammation and immune dysregulation differ by sex. In turn, this may aid in the pursuit of discovering sex-specific biomarkers to identify obese individuals at particular risk of heart failure.

Abstract C003: Multimodal and multiplexed spatial regulation of the breast collagen proteome: Comparative analysis in Black and White women from normal breast tissue and triple-negative breast cancer

Abstract Non-Hispanic Black women (BW) are diagnosed with more aggressive breast cancer subtypes at younger ages and have higher mortality rates compared to non-Hispanic White women (WW). Within the breast microenvironment emergent data shows that stromal collagen plays a significant role in aggressiveness of breast cancer linked to ancestral traits. However, the molecular composition of collagen stroma has yet to be linked to ancestry and aggressiveness of breast cancer. The purpose of this study was to evaluate the microenvironment within ancestry-defined normal breast and triple-negative breast cancer (TNBC) to understand spatial regulation of collagen stroma that may link to ancestral differences in cancer. Multimodal, multiplexed spatial analyses were performed. Techniques included Second Harmonic Generation (SHG) to measure collagen fibers, mass spectrometry imaging to measure the collagen proteome, and mass-tagged antibody panels to investigate immune signatures in TNBC. Normal breast tissues were obtained from the Susan G. Komen Tissue Bank, African American (AA; n=20) and European American (EA; n=20) samples were annotated with risk scores, age, Body Mass Index (BMI), Breast Imaging-Reporting and Data System (BI-RADS), continental ancestry, and family history. Triple-negative breast cancer (TNBC, n=80) tissues from BW with extensive clinical annotations including age, BMI, stage, metastasis, and survival were evaluated in comparison to 20+ TNBC tissues from WW. In the normal breast cohort, evaluation of BMI (overweight and obese) within ancestry groups showed significant but opposing differences in fiber width measurements. As BMI increases, AA showed statistically thicker fibers while EA showed statistically thinner fibers. At the protein level, two types of collagens were significantly higher in EA and one type is higher in AA. Within the TNBC cohort, collagen fiber length and width measurements by SHG were significantly shorter and thinner in stage II compared to stage I but no other stage comparisons were significant. This may indicate that ECM remodeling occurs at earlier stages in TNBC. Initial analyses showed that collagen peptide intensities significantly vary by TNBC stage (75 peptides, ANOVA test FDR p-value < 0.05). In the cohort of women who survived, 90 ECM peptides significantly correlated with collagen fiber widths, indicating a relationship between collagen physical and molecular properties within a defined region. Seven immune markers defined differences in survival when grouped by high vs low expression within tumor regions. These markers include CD20 (Hazard Ratio HR = 7.99, p-value = 0.002) and PD-1 (HR = 3.68, p-value = 0.048), which have both been studied in breast cancer. Current work focuses on collagen peptide post-translational modifications that may differ in TNBC patients by vital status and are detected in normal breast at risk of cancer. These findings support that molecular composition in breast stroma contributes to aggressive ancestral traits of breast cancer risk and triple-negative breast cancer. Citation Format: Jaclyn B. Dunne, Heather Jensen-Smith, Laura Spruill, Taylor S. Hulahan, Jade K. Macdonald, George E. Sandusky, M. A. Hollingsworth, Anand S. Mehta, Richard R. Drake, Jeffrey R. Marks, Harikrishna Nakshatri, Marvella E. Ford, Peggi M. Angel. Multimodal and multiplexed spatial regulation of the breast collagen proteome: Comparative analysis in Black and White women from normal breast tissue and triple-negative breast cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C003.

High glucose induces DNA methyltransferase 1 dependent epigenetic reprogramming of the endothelial exosome proteome in type 2 diabetes

In response to hyperglycemia, endothelial cells (ECs) release exosomes with altered protein content and contribute to paracrine signalling, subsequently leading to vascular dysfunction in type 2 diabetes (T2D). High glucose reprograms DNA methylation patterns in various cell/tissue types, including ECs, resulting in pathologically relevant changes in cellular and extracellular proteome. However, DNA methylation-based proteome reprogramming in endothelial exosomes and associated pathological implications in T2D are not known. Hence, in the present study, we used Human umbilical vein endothelial cells (HUVECs), High Fat Diet (HFD) induced diabetic mice (C57BL/6) and clinical models to understand epigenetic basis of exosome proteome regulation in T2D pathogenesis . Exosomes were isolated by size exclusion chromatography and subjected to tandem mass tag (TMT) labelled quantitative proteomics and bioinformatics analysis. Immunoblotting was performed to validate exosome protein signature in clinically characterized individuals with T2D. We observed ECs cultured in high glucose and aortic ECs from HFD mouse expressed elevated DNA methyltransferase1 (DNMT1) levels. Quantitative proteomics of exosomes isolated from ECs treated with high glucose and overexpressing DNMT1 showed significant alterations in both protein levels and post translational modifications which were aligned to T2D associated vascular functions. Based on ontology and gene-function-disease interaction analysis, differentially expressed exosome proteins such as Thrombospondin1, Pentraxin3 and Cystatin C related to vascular complications were significantly increased in HUVECs treated with high glucose and HFD animals and T2D individuals with higher levels of glycated hemoglobin. These proteins were reduced upon treatment with 5-Aza-2’-deoxycytidine. Our study shows epigenetic regulation of exosome proteome in T2D associated vascular complications.

Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics.

Ductal carcinoma in situ (DCIS) is a heterogeneous breast disease that remains challenging to treat due to its unpredictable progression to invasive breast cancer (IBC). Contemporary literature has become increasingly focused on extracellular matrix (ECM) alterations with breast cancer progression. However, the spatial regulation of the ECM proteome in DCIS has yet to be investigated in relation to IBC. We hypothesized that DCIS and IBC present distinct ECM proteomes that could discriminate between these pathologies. Tissue sections of pure DCIS, mixed DCIS-IBC, or pure IBC (n = 22) with detailed pathological annotations were investigated by multiplexed spatial proteomics. Across tissues, 1,005 ECM peptides were detected in pathologically annotated regions and their surrounding extracellular microenvironments. A comparison of DCIS to IBC pathologies demonstrated 43 significantly altered ECM peptides. Notably, eight fibrillar collagen peptides could distinguish with high specificity and sensitivity between DCIS and IBC. Lesion-targeted proteomic imaging revealed heterogeneity of the ECM proteome surrounding individual DCIS lesions. Multiplexed spatial proteomics reported an invasive cancer field effect, in which DCIS lesions in closer proximity to IBC shared a more similar ECM profile to IBC than distal counterparts. Defining the ECM proteomic microenvironment provides novel molecular insights relating to DCIS and IBC.

Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions

Local mRNA translation in axons is critical for the spatiotemporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons; however, how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing rat hippocampal cultured neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER-ribosome interactions causes defects in axon morphology. Our results establish a role for the axonal ER in dynamically localizing mRNA translation, which is important for proper neuron development.

DNA methyltransferase isoforms regulate endothelial cell exosome proteome composition

Extrinsic and intrinsic pathological stimuli in vascular disorders induce DNA methylation based epigenetic reprogramming in endothelial cells, which leads to perturbed gene expression and subsequently results in endothelial dysfunction (ED). ED is also characterized by release of exosomes with altered proteome leading to paracrine interactions in vasculature and subsequently contributing to manifestation, progression and severity of vascular complications. However, epigenetic regulation of exosome proteome is not known. Hence, our present study aimed to understand influence of DNA methylation on exosome proteome composition and their influence on endothelial cell (EC) function. DNMT isoforms (DNMT1, DNMT3A, and DNMT3B) were overexpressed using lentivirus in ECs. Exosomes were isolated and characterized from ECs overexpressing DNMT isoforms and C57BL/6 mice plasma treated with 5-aza-2′-deoxycytidine. 3D spheroid assay was performed to understand the influence of exosomes derived from cells overexpressing DNMTs on EC functions. Further, the exosomes were subjected to TMT labelled proteomics analysis followed by validation. 3D spheroid assay showed increase in the pro-angiogenic activity in response to exosomes derived from DNMT overexpressing cells which was impeded by inclusion of 5-aza-2′-deoxycytidine. Our results showed that exosome proteome and PTMs were significantly modulated and were associated with dysregulation of vascular homeostasis, metabolism, inflammation and endothelial cell functions. In vitro and in vivo validation showed elevated DNMT1 and TGF-β1 exosome proteins due to DNMT1 and DNMT3A overexpression, but not DNMT3B which was mitigated by 5-aza-2′-deoxycytidine indicating epigenetic regulation. Further, exosomes induced ED as evidenced by reduced expression of phospho-eNOSser1177. Our study unveils epigenetically regulated exosome proteins, aiding management of vascular complications.

Genetic regulation of human brain proteome reveals proteins implicated in psychiatric disorders.

Psychiatric disorders are highly heritable yet polygenic, potentially involving hundreds of risk genes. Genome-wide association studies have identified hundreds of genomic susceptibility loci with susceptibility to psychiatric disorders; however, the contribution of these loci to the underlying psychopathology and etiology remains elusive. Here we generated deep human brain proteomics data by quantifying 11,608 proteins across 268 subjects using 11-plex tandem mass tag coupled with two-dimensional liquid chromatography-tandem mass spectrometry. Our analysis revealed 788 cis-acting protein quantitative trait loci associated with the expression of 883 proteins at a genome-wide false discovery rate <5%. In contrast to expression at the transcript level and complex diseases that are found to be mainly influenced by noncoding variants, we found protein expression level tends to be regulated by non-synonymous variants. We also provided evidence of 76 shared regulatory signals between gene expression and protein abundance. Mediation analysis revealed that for most (88%) of the colocalized genes, the expression levels of their corresponding proteins are regulated by cis-pQTLs via gene transcription. Using summary data-based Mendelian randomization analysis, we identified 4 proteins and 19 genes that are causally associated with schizophrenia. We further integrated multiple omics data with network analysis to prioritize candidate genes for schizophrenia risk loci. Collectively, our findings underscore the potential of proteome-wide linkage analysis in gaining mechanistic insights into the pathogenesis of psychiatric disorders.

Mechanism of Decision Making between Autophagy and Apoptosis Induction upon Endoplasmic Reticulum Stress.

Dynamic regulation of the cellular proteome is mainly controlled in the endoplasmic reticulum (ER). Accumulation of misfolded proteins due to ER stress leads to the activation of unfolded protein response (UPR). The primary role of UPR is to reduce the bulk of damages and try to drive back the system to the former or a new homeostatic state by autophagy, while an excessive level of stress results in apoptosis. It has already been proven that the proper order and characteristic features of both surviving and self-killing mechanisms are controlled by negative and positive feedback loops, respectively. The new results suggest that these feedback loops are found not only within but also between branches of the UPR, fine-tuning the response to ER stress. In this review, we summarize the recent knowledge of the dynamical characteristic of endoplasmic reticulum stress response mechanism by using both theoretical and molecular biological techniques. In addition, this review pays special attention to describing the mechanism of action of the dynamical features of the feedback loops controlling cellular life-and-death decision upon ER stress. Since ER stress appears in diseases that are common worldwide, a more detailed understanding of the behaviour of the stress response is of medical importance.

Synergistic effect of sildenafil combined with controlled hypothermia to alleviate microglial activation after neonatal hypoxia–ischemia in rats

Background and purposeThe only validated treatment to prevent brain damage associated with hypoxia–ischemia (HI) encephalopathy of the newborn is controlled hypothermia with limited benefits. Additional putative neuroprotective drug candidates include sildenafil citrate, a phosphodiesterase-type 5 inhibitor. The main objective of this preclinical study is to assess its ability to reduce HI-induced neuroinflammation, in particular through its potential effect on microglial activation.MethodsHI was induced in P10 Sprague–Dawley rats by unilateral carotid permanent artery occlusion and hypoxia (HI) and treated by either hypothermia (HT) alone, Sildenafil (Sild) alone or combined treatment (SildHT). Lesion size and glial activation were analyzed by immunohistochemistry, qRT-PCR, and proteomic analyses performed at P13.ResultsNone of the treatments was associated with a significant early reduction in lesion size 72h after HI, despite significant changes in tissue loss distribution. Significant reductions in both Iba1 + (within the ipsilateral hemisphere) and GFAP + cells (within the ipsilateral hippocampus) were observed in SildHT group, but not in the other treatment groups. In microglia-sorted cells, pro-inflammatory markers, i.e. Il1b, Il6, Nos2, and CD86 were significantly downregulated in SildHT treatment group only. These changes were restricted to the ipsilateral hemisphere, were not evidenced in sorted astrocytes, and were not sex dependent. Proteomic analyses in sorted microglia refined the pro-inflammatory effect of HI and confirmed a biologically relevant impact of SildHT on specific molecular pathways including genes related to neutrophilic functions.ConclusionsOur findings suggest that Sildenafil combined with controlled hypothermia produces maximum effect in mitigating microglial activation induced by HI through complex proteomic regulation. The reduction of neuroinflammation induced by Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.

Dehydration-responsive cytoskeleton proteome of rice reveals reprograming of key molecular pathways to mediate metabolic adaptation and cell survival

The plant cytoskeletal proteins play a key role that control cytoskeleton dynamics, contributing to crucial biological processes such as cell wall morphogenesis, stomatal conductance and abscisic acid accumulation in repercussion to water-deficit stress or dehydration. Yet, it is still completely unknown which specific biochemical processes and regulatory mechanisms the cytoskeleton uses to drive dehydration tolerance. To better understand the role of cytoskeleton, we developed the dehydration-responsive cytoskeletal proteome map of a resilient rice cultivar. Initially, four-week-old rice plants were exposed to progressive dehydration, and the magnitude of dehydration-induced compensatory physiological responses was monitored in terms of physicochemical indices. The organelle fractionation in conjunction with label-free quantitative proteome analysis led to the identification of 955 dehydration-responsive cytoskeletal proteins (DRCPs). To our knowledge, this is the first report of a stress-responsive plant cytoskeletal proteome, representing the largest inventory of cytoskeleton and cytoskeleton-associated proteins. The DRCPs were apparently involved in a wide array of intra-cellular molecules transportation, organelles positioning, cytoskeleton organization followed by different metabolic processes including amino acid metabolism. These findings presented open a unique view on global regulation of plant cytoskeletal proteome is intimately linked to cellular metabolic rewiring of adaptive responses, and potentially confer dehydration tolerance, especially in rice, and other crop species, in general.

Comparative Analysis of Differential Cellular Transcriptome and Proteome Regulation by HIV-1 and HIV-2 Pseudovirions in the Early Phase of Infection.

In spite of the similar structural and genomic organization of human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), striking differences exist between them in terms of replication dynamics and clinical manifestation of infection. Although the pathomechanism of HIV-1 infection is well characterized, relatively few data are available regarding HIV-2 viral replication and its interaction with host-cell proteins during the early phase of infection. We utilized proteo-transcriptomic analyses to determine differential genome expression and proteomic changes induced by transduction with HIV-1/2 pseudovirions during 8, 12 and 26 h time-points in HEK-293T cells. We show that alteration in the cellular milieu was indeed different between the two pseudovirions. The significantly higher number of genes altered by HIV-2 in the first two time-points suggests a more diverse yet subtle effect on the host cell, preparing the infected cell for integration and latency. On the other hand, GO analysis showed that, while HIV-1 induced cellular oxidative stress and had a greater effect on cellular metabolism, HIV-2 mostly affected genes involved in cell adhesion, extracellular matrix organization or cellular differentiation. Proteomics analysis revealed that HIV-2 significantly downregulated the expression of proteins involved in mRNA processing and translation. Meanwhile, HIV-1 influenced the cellular level of translation initiation factors and chaperones. Our study provides insight into the understudied replication cycle of HIV-2 and enriches our knowledge about the use of HIV-based lentiviral vectors in general.

Investigating genomic, proteomic, and post-transcriptional regulation profiles in colorectal cancer: a comparative study between primary tumors and associated metastases

IntroductionApproximately 50% of patients with primary colorectal carcinoma develop liver metastases. This study investigates the possible molecular discrepancies between primary colorectal cancer (pCRC) and their respective metastases.MethodsA total of 22 pairs of pCRC and metastases were tested. Mutation profiling of 26 cancer-associated genes was undertaken in 22/22primary-metastasis tumour pairs using next-generation sequencing, whilst the expression of a panel of six microRNAs (miRNAs) was investigated using qPCRin 21/22 pairs and 22 protein biomarkers was tested using Reverse Phase Protein Array (RPPA)in 20/22 patients’ tumour pairs.ResultsAmong the primary and metastatic tumours the mutation rates for the individual genes are as follows:TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. The primary-metastasis tumour mutation status was identical in 54/60 (90%) loci. However, there was discordance in heterogeneity status in 40/58 genetic loci (z-score = 6.246, difference = 0.3793, P < 0.0001). Furthermore, there was loss of concordance in miRNA expression status between primary and metastatic tumours, and 57.14–80.95% of the primary-metastases tumour pairs showed altered primary-metastasis relative expression in all the miRNAs tested. Moreover, 16 of 20 (80%) tumour pairs showed alteration in at least 3 of 6 (50%) of the protein biomarker pathways analysed.ConclusionThe molecular alterations of primary colorectal tumours differ significantly from those of their matched metastases. These differences have profound implications for patients’ prognoses and response to therapy.

Different UV-B exposure times induce phenotypic, transcriptomic, and proteomic changes in wine grape leaves

The Shangri-La region, located in southwest China, is known for its dry climate and intense UltraViolet (UV) radiation due to its high altitude. Research conducted in the past has demonstrated that UV radiation, particularly UV-B radiation, enhances plants' ability to withstand cold temperatures, diseases, and arid conditions. Indeed, the Cabernet Sauvignon grape variety, predominantly cultivated in Shangri-La where the UV intensity reaches 21–22w/m2, has displayed remarkable resilience in recent decades. However, the specific impact of UV-B radiation on Cabernet Sauvignon grape leaves has yet to be fully understood. Consequently, our objective is to investigate the influence of UV radiation on grapes grown at high altitudes. To gain insights into the short-term effects on grape leaves, we conducted a comprehensive investigation involving anatomical and photosynthetic changes. Additionally, we analyzed the transcriptomic and proteomic regulation associated with these changes following exposure to UV-B radiation at intensities ranging from 15–20w/m2 for durations of 3 and 7 days.The findings of our study demonstrated that exposure to UV-B radiation, particularly at intensities surpassing 10w/m2, led to noticeable changes in the morphology of epidermal and palisade tissue cells. Furthermore, after a 3-day exposure to UV-B, we observed an upregulation of proteins responsible for cytoskeletal organization and cell wall synthesis. Conversely, following a 7-day treatment, proteins associated with disease resistance and antioxidants showed increased expression. Transcriptomic analysis revealed a significant enrichment of proteins and genes linked to pathogen infection, defense mechanisms, and antioxidant processes. Overall, our research highlighted the positive impact of intense UV radiation at high altitudes on enhancing disease resistance and environmental adaptability in Cabernet Sauvignon grape plants.

Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome.

Substance use disorders are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based quantitative proteomics, along with a data-independent acquisition analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveal cocaine and morphine differentially alter diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between the phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome. The proteomics data in this study are available via ProteomeXchange with identifier PXD042043.

Mapping early serum proteome signatures of liver regeneration in living donor liver transplant cases.

The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.

Proteomic reconfigurations underlying physiological alterations in poplar roots in acclimation to changing nitrogen availability

Although the roots of woody plants are essential for nitrogen (N) uptake and assimilation, proteomic regulation underlying these processes is unclear in acclimation to changing N availability. Here, we carried out physiological and proteomic assays in Populus × canescens sapling roots treated with one of low, normal and high levels of NH4NO3. N deficiency induced lower concentrations of NH4+, total N, amino acids, inhibited enzymatic activities of nitrate reductase and glutamate synthase, decreased levels of abscisic acid and salicylic acid in poplar roots, and high N had the opposite effects. There were 552 and 247 significantly differentially abundant proteins (DAPs) in low and high vs normal N-supplied poplar roots, respectively. Notably, the abundances of a few poplar proteins, including nitrite reductase 1, glutamate synthase 1, and glutamate dehydrogenase 1, corresponded well to N-induced physiological changes in poplar roots. Functional characterization of N-responsive poplar ammonium transporter 2;1 and glutamate dehydrogenase 2 suggests both play pivotal roles in N uptake and assimilation in response to altering N levels. These results suggest that the growth, N uptake and assimilation, and phytohormonal levels are inhibited by N deficiency, but stimulated by high N, and the proteomic reconfigurations occur underlying these physiological alterations in poplars in acclimation to changing N availability.

Abstract A031: KRAS (G12C) mediated mRNA translation program

Abstract KRAS-dependent activation of mRNA translation signaling is a common feature in cancer however, the KRAS-specific translation landscape and functional proteome are not fully characterized in a genome-wide manner. We show that mutant KRAS drives specific mechanisms of translational control to support cancer growth. We have utilized the technology of ribosome footprinting combined with advanced sequencing methodology to define the translation landscape affected by a specific inhibitor of KRAS, sotorasib that targets G12C mutant KRAS activity. We show that acute treatment of sotorasib inhibits the translation of key oncogenic proteins including MYC and MYC targets. KRAS (G12C) inhibition profoundly affects the translation of ribosomal proteins and translation elongation factors. G12C mutant KRAS remarkably affects the translation of proteins involved in the ROS and DNA repair pathways in cancer. Moreover, these translational changes are mediated through specific RNA motifs and RNA binding proteins. In summary, our study defines the KRAS (G12C) specific regulation of translation and provides the mechanism of KRAS (G12C) mediated regulation of cancer proteome. Citation Format: Kamini Singh. KRAS (G12C) mediated mRNA translation program [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A031.

Tailored Apoptotic Vesicle Delivery Platform for Inflammatory Regulation and Tissue Repair to Ameliorate Ischemic Stroke

Apoptotic vesicles (ApoVs) hold great promise for inflammatory regulation and tissue repair. However, little effort has been dedicated to developing ApoV-based drug delivery platforms, while the insufficient targeting capability of ApoVs also limits their clinical applications. This work presents a platform architecture that integrates apoptosis induction, drug loading, and functionalized proteome regulation, followed by targeting modification, enabling the creation of an apoptotic vesicle delivery system to treat ischemic stroke. Briefly, α-mangostin (α-M) was utilized to induce mesenchymal stem cell (MSC) apoptosis while being loaded onto MSC-derived ApoVs as an anti-oxidant and anti-inflammatory agent for cerebral ischemia/reperfusion injury. Matrix metalloproteinase activatable cell-penetrating peptide (MAP), a microenvironment-responsive targeting peptide, was modified on the surface of ApoVs to obtain the MAP-functionalized α-M-loaded ApoVs. Such engineered ApoVs targeted the injured ischemic brain after systemic injection and achieved an enhanced neuroprotective activity due to the synergistic effect of ApoVs and α-M. The internal protein payloads of ApoVs, upon α-M activation, were found engaged in regulating immunological response, angiogenesis, and cell proliferation, all of which contributed to the therapeutic effects of ApoVs. The findings provide a universal framework for creating ApoV-based therapeutic drug delivery systems for the amelioration of inflammatory diseases and demonstrate the potential of MSC-derived ApoVs to treat neural injury.

Higher‐order modular regulation of the human proteome

Operons are transcriptional modules that allow bacteria to adapt to environmental changes by coordinately expressing the relevant set of genes. In humans, biological pathways and their regulation are more complex. If and how human cells coordinate the expression of entire biological processes is unclear. Here, we capture 31 higher‐order co‐regulation modules, which we term progulons, by help of supervised machine‐learning on proteomics data. Progulons consist of dozens to hundreds of proteins that together mediate core cellular functions. They are not restricted to physical interactions or co‐localisation. Progulon abundance changes are primarily controlled at the level of protein synthesis and degradation. Implemented as a web app at www.proteomehd.net/progulonFinder, our approach enables the targeted search for progulons of specific cellular processes. We use it to identify a DNA replication progulon and reveal multiple new replication factors, validated by extensive phenotyping of siRNA‐induced knockdowns. Progulons provide a new entry point into the molecular understanding of biological processes.

Abstract P2-21-03: Unique Collagen Peptide Signatures between Ductal Carcinoma in Situ and Invasive Breast Cancer by Mass Spectrometry Tissue Imaging

Abstract Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). Recent publications have implicated the crucial role of the tumor microenvironment particularly stromal differences between DCIS patients who progress to IBC (progressors) and those that do not (non-progressors). However, spatial regulation of the collagen proteome has yet to be investigated in the context of disease progression in DCIS. In this study, we hypothesized that the collagen proteome was significantly altered between DCIS and IBC and that differentiating collagen peptide signatures could be related to clinical outcomes. Our initial studies investigated collagen peptide signatures in lumpectomies (n=13) annotated as DCIS, DCIS and invasive ductal carcinoma (IDC), or IDC only. We leveraged our previously published method for spatial imaging of collagen proteomics on tissue to report collagen types and collagen post-translational modifications including 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Over 1000 peaks were found to be linked to annotated pathologies or adjacent regions. Initial comparison of DCIS to IDC lesions demonstrated 63 differentially expressed peaks between these regions by unpaired, two-tailed t-test (p&amp;lt; 0.001). Image segmentation of the 315,541 pixels demonstrated 16 high-level hierarchical groups designating unique spatially localized ECM proteomic groups. Notably, these groups overlaid with histopathological features and pathological annotations. Next, we investigated collagen peptide signatures in a subset of DCIS samples from the Resource of Archival Breast Tissue (RAHBT) (n=37). Samples were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. In our preliminary analysis, we found two peptide peaks that could distinguish the solid subtype (n=22) from comedo necrosis (n=4) and one peak that could discriminate between the cribriform (n=8) and solid subtype (n=22) by area under the receiver operating curve (AUROC)≥0.75 and Wilson/Brown t-test (p&amp;lt; 0.05). Evaluated per clinical outcome, four ECM peptides showed significantly different peak intensities in progressors with IBC recurrence (n=7) compared to non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p&amp;lt; 0.05). One peptide had significantly different peak intensities between progressors with contralateral IBC recurrence and DCIS recurrence (n=5) and non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p&amp;lt; 0.05). Overall, the data suggest that unique collagen signatures in DCIS could be useful for understanding recurrence and progression to IBC. Further investigation of the spatial distribution of the collagen proteome within DCIS pathologies and relative to clinical outcome is warranted. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Graham A. Colditz, E Shelley Hwang, Robert West, Laura Spruill, Jeffrey Marks, Richard R. Drake, Peggi M. Angel. Unique Collagen Peptide Signatures between Ductal Carcinoma in Situ and Invasive Breast Cancer by Mass Spectrometry Tissue Imaging [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-03.