Significant Changes In Protein Expression Research Articles

Comparative proteomics reveals different protein expression in platelets in patients with alcoholic liver cirrhosis

BackgroundThe role of platelets in disease progression as well as the function of platelets as part of the haemostatic and immunological system in patients with liver cirrhosis is only incompletely understood. This is partly due to difficulties in assessing platelet function. Proteome analyses of platelets have been used to further investigate the role of platelets in other diseases.AimTo assess possible changes in the platelet proteome during different stages of alcohol induced liver cirrhosis compared to healthy donors.Patients and methodsA 45 ml blood sample was drawn from 18 participants aged 18–80 years evenly divided into three groups of healthy donors, patients with less advanced alcohol induced liver cirrhosis (Child-Pugh < 7) and patients with advanced liver cirrhosis (Child-Pugh > 10). The blood was processed to isolate platelets and perform subsequent two-dimensional gel-electrophoresis using a SYPRO™ Ruby dye. After computational analysation significantly in- or decreased protein spots (defined as a two-fold abundance change between different study cohorts and ANOVA < 0.05) were identified via liquid chromatography–mass spectrometry (LCMS) and searching against human protein databases.ResultsThe comparative analysis identified four platelet proteins with progressively decreased protein expression in patients with liver cirrhosis. More specifically Ras-related protein Rab-7a (Rab-7a), Ran-specific binding protein 1 (RANBP1), Rho GDP-dissociation inhibitor 1 (RhoGDI1), and 14–3-3 gamma.ConclusionThere is significant change in protein expression in the platelet proteome throughout the disease progression of alcohol induced liver cirrhosis. The identified proteins are possibly involved in haemostatic and immunoregulatory function of platelets.

Abstract 18558: Modeling Cardiac Amyloidosis: Insights Into Pathogenesis and Therapeutic Strategies

Introduction: Cardiac transthyretin (TTR) amyloidosis is an underdiagnosed cause of heart failure and cardiac arrhythmias. The lack of relevant human cardiac tissue models hinders understanding of the underlying mechanisms and development of effective treatments. Hypothesis: We aimed to establish an in vitro model of cardiac amyloidosis using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) and aggregated TTR and investigate cellular changes and potential therapeutic targets. Methods: Wild-type and mutated TTR proteins were aggregated and added to hiPSC-CM cultures directly or embedded in Matrigel used for culture-plate coating. Single-cell and two- and three-dimensional hiPSC-CM models were used for real-time microscopy, calcium and voltage imaging, immunostaining, apoptosis, ROS assays, and proteomics studies with mass spectrometry. Results: Immunohistochemical and histological staining confirmed the deposition of TTR aggregates within the hiPSC-CM tissue models. Cells exposed to TTR aggregates exhibited increased apoptosis and increased production of reactive oxygen species (ROS). TTR-treated cells also displayed irregular calcium transients, characterized by oscillating calcium peaks and double-humped signals. The presence of TTR also induced morphological changes, including cell shrinkage at the single-cell level and the formation of holes in 2D hiPSC-CM cell sheets due to cell migration and apoptosis. These holes led to conduction abnormalities and the development of reentrant arrhythmias, specifically spiral waves. The severity of these abnormalities was markedly increased when mutant TTR aggregates were used. Proteomics studies suggest a potential involvement of the mTOR signaling pathway in the pathogenesis of cardiac amyloidosis. Conclusions: Our in vitro model successfully recapitulated key features of cardiac amyloidosis and revealed significant protein expression changes. These findings provide valuable insights into disease pathogenesis and potential therapeutic targets and highlight the utility of our model for studying cardiac amyloidosis for the development and evaluation of novel therapeutic strategies.

Regulation of Expression of Extracellular Matrix Proteins by Differential Target Multiplexed Spinal Cord Stimulation (SCS) and Traditional Low-Rate SCS in a Rat Nerve Injury Model.

There is limited research on the association between the extracellular matrix (ECM) and chronic neuropathic pain. The objective of this study was twofold. Firstly, we aimed to assess changes in expression levels and the phosphorylation of ECM-related proteins due to the spared nerve injury (SNI) model of neuropathic pain. Secondly, two modalities of spinal cord stimulation (SCS) were compared for their ability to reverse the changes induced by the pain model back toward normal, non-injury levels. We identified 186 proteins as ECM-related and as having significant changes in protein expression among at least one of the four experimental groups. Of the two SCS treatments, the differential target multiplexed programming (DTMP) approach reversed expression levels of 83% of proteins affected by the pain model back to levels seen in uninjured animals, whereas a low-rate (LR-SCS) approach reversed 67%. There were 93 ECM-related proteins identified in the phosphoproteomic dataset, having a combined 883 phosphorylated isoforms. DTMP back-regulated 76% of phosphoproteins affected by the pain model back toward levels found in uninjured animals, whereas LR-SCS back-regulated 58%. This study expands our knowledge of ECM-related proteins responding to a neuropathic pain model as well as providing a better perspective on the mechanism of action of SCS therapy.

Investigating serum extracellular vesicles in Cystic Fibrosis

BackgroundExtracellular vesicles (EVs) are emerging as biomarkers of disease with diagnostic potential in CF. With the advent of highly effective modulator therapy, sputum production is less common and there is a need to identify novel markers of CF disease progression, exacerbation and response to therapies in accessible fluids such as serum. MethodsWe used size exclusion chromatography (SEC) to isolate and characterise EVs from the blood of PWCF of different ages and compared to ultracentrifugation (UC). We used nanoparticle tracking analysis to measure the number of EVs present in serum obtained from children and adults with CF. Mass spectrometry based proteomics was used to characterise protein expression changes between the groups. ResultsEVs were successfully isolated in SEC fractions from 250 µl serum from PWCF in greater numbers (p <0.01) than density ultracentrifugation. There was not a significant difference in EV numbers between young children with CF and controls. However, there was significantly more EVs in adults compared to children (<6yrs) (p < 0.05). EVs from PWCF before and after Kaftrio treatment were also analysed. Significant protein expression changes were observed within all 3 group. The largest changes detected were between children and adults with CF (57 proteins had a 1.5 fold change in expression with 19 significant changes p < 0.05) and PWCF taking Kaftrio (24 significant changes in EV protein expression was observed 12 months post treatment). ConclusionIn this pilot study, we performed an initial characterisation of EVs in serum from PWCF demonstrating the potential of serum EVs for further diagnostic investigation.

On the Feasibility of Using an Ultra-Fast DirectMS1 Method of Proteome-Wide Analysis for Searching Drug Targets in Chemical Proteomics

Protein quantitation in tissue cells or physiological fluids based on liquid chromatography/mass spectrometry is one of the key sources of information on the mechanisms of cell functioning during chemotherapeutic treatment. Information on significant changes in protein expression upon treatment can be obtained by chemical proteomics and requires analysis of the cellular proteomes, as well as development of experimental and bioinformatic methods for identification of the drug targets. Low throughput of whole proteome analysis based on liquid chromatography and tandem mass spectrometry is one of the main factors limiting the scale of these studies. The method of direct mass spectrometric identification of proteins, DirectMS1, is one of the approaches developed in recent years allowing ultrafast proteome-wide analyses employing minute-scale gradients for separation of proteolytic mixtures. Aim of this work was evaluation of both possibilities and limitations of the method for identification of drug targets at the level of whole proteome and for revealing cellular processes activated by the treatment. Particularly, the available literature data on chemical proteomics obtained earlier for a large set of onco-pharmaceuticals using multiplex quantitative proteome profiling were analyzed. The results obtained were further compared with the proteome-wide data acquired by the DirectMS1 method using ultrashort separation gradients to evaluate efficiency of the method in identifying known drug targets. Using ovarian cancer cell line A2780 as an example, a whole-proteome comparison of two cell lysis techniques was performed, including the freeze-thaw lysis commonly employed in chemical proteomics and the one based on ultrasonication for cell disruption, which is the widely accepted as a standard in proteomic studies. Also, the proteome-wide profiling was performed using ultrafast DirectMS1 method for A2780 cell line treated with lonidamine, followed by gene ontology analyses to evaluate capabilities of the method in revealing regulation of proteins in the cellular processes associated with drug treatment.

Skin proteomic analysis of immune activation associated with regression of melanoma metastases induced by diphencyprone

To the Editor: Diphencyprone (DPCP), a hapten that causes delayed-type hypersensitivity reactions, has been used to treat warts, alopecia areata, and cutaneous metastases in melanoma patients.1Damian D.L. Saw R.P. Thompson J.F. Topical immunotherapy with diphencyprone for in transit and cutaneously metastatic melanoma.J Surg Oncol. 2014; 109: 308-313https://doi.org/10.1002/jso.23506Crossref PubMed Scopus (51) Google Scholar Previously, our group performed transcriptomic analysis of skin biopsies from melanoma metastases treated with topical DPCP, revealing significant increases in Th1-related genes but not in genes of other major T-cell subsets.2Gulati N. Garcet S. Fuentes-Duculan J. et al.Molecular profiling of immune activation associated with regression of melanoma metastases induced by diphencyprone.J Invest Dermatol. 2016; 136: 2101-2103https://doi.org/10.1016/j.jid.20 16.06.611Crossref PubMed Scopus (0) Google Scholar While gene expression profiling has long been used as a proxy for protein expression, correlation between messenger RNA (mRNA) and protein levels is inconsistent due to factors such as post-transcriptional regulation. Therefore, to more precisely ascertain the mechanisms involved in immune-mediated tumor regression induced by DPCP, we performed proteomic analysis of skin biopsies from 5 patients with cutaneous melanoma metastases treated with DPCP twice weekly for 7 to 14 weeks (Supplemental Material and Methods and Supplementary Table I, available via Mendeley at https://doi.org/10.17632/f5ttw4mygy.1). In this study, we assessed 96 proteins using the Olink immuno-oncology panel for the 5 patients who underwent molecular profiling. All patients had expected skin inflammation and at least partial regression of skin metastases, without systemic side effects. There were 10 proteins (interleukin 6 [IL-6], IL-12, IL-18, TIE2, HGF, PDGFB, TNFSF14, PD-L2, MMP7, MMP12) significantly upregulated (P < .05) in post-treatment versus pretreatment metastases (Fig 1), including biomarkers associated with Th1 response (IL-12), innate immunity (IL-6), and immune checkpoints (PD-L2) (P < .05). All of these proteins had progressively increased expression in cutaneous metastases with repeated DPCP applications, and similar increases were observed in nonlesional skin of these patients following a single DPCP application. Prior work has shown that administration of IL-18 in mice increases activated T cells and natural killer cells that contribute to effective antimelanoma immunity,3Zhou T. Damsky W. Weizman O.E. et al.IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy.Nature. 2020; 583: 609-614https://doi.org/10.1038/s41586-020-2422-6Crossref PubMed Scopus (93) Google Scholar so the significant upregulation of this protein in our study may contribute to the successful skin metastasis regression observed in our patients. TNFSF14 also likely plays an active role in immune-mediated regression, as in vivo studies have demonstrated an association between the expression of this protein in melanoma metastasis lesions and proliferation of T cells.4Mortarini R. Scarito A. Nonaka D. et al.Constitutive expression and costimulatory function of LIGHT/TNFSF14 on human melanoma cells and melanoma-derived microvesicles.Cancer Res. 15 2005; 65: 3428-3436https://doi.org/10.1158/0008-5472.CAN-04-3239Crossref PubMed Scopus (35) Google Scholar Additionally, there were 40 proteins significantly upregulated in pretreatment metastases versus untreated nonlesional skin (P < .05) (Fig 1), suggesting baseline immune activation in melanoma metastases. Upon DPCP treatment, changes in nonlesional skin positively correlated with those in skin metastases (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/f5ttw4mygy.1; Spearman correlation = 0.61, P < .0001). We performed a correlation analysis between the proteomic data and the previously published mRNA data,2Gulati N. Garcet S. Fuentes-Duculan J. et al.Molecular profiling of immune activation associated with regression of melanoma metastases induced by diphencyprone.J Invest Dermatol. 2016; 136: 2101-2103https://doi.org/10.1016/j.jid.20 16.06.611Crossref PubMed Scopus (0) Google Scholar which both were completed on the same tissue samples, to assess the relationship between these 2 “omic” approaches (Fig 2). Correlation was poor (r = −0.1, P = .4012), including for proteins that were significantly upregulated by proteomics (IL-6 and TNFSF14) (both P < .05). Gene expression from mRNA is variably correlated with protein expression, the latter being influenced by many regulatory processes prior to translation. Another explanation for the discrepancy observed between mRNA and protein expression includes the shorter half-life of mRNA molecules compared to proteins.5Mauger D.M. Cabral B.J. Presnyak V. et al.mRNA structure regulates protein expression through changes in functional half-life.Proc Natl Acad Sci U S A. 2019; 116: 24075-24083https://doi.org/10.1073/pnas.1908052116Crossref PubMed Scopus (152) Google Scholar Despite our limited sample size, we were able to observe statistically significant protein expression changes, and this study is, to our knowledge, the first to assess the proteomic signature of successful immune-mediated regression of cutaneous melanoma metastases induced by a topical immunomodulator. Given the lack of correlation between mRNA and protein expression, along with the relatively greater feasibility of measuring protein compared to gene expression levels, our study demonstrates that proteomic analysis can give unique and potentially complementary insights into immune reactions induced by topical immunotherapy. None disclosed.

Comparative proteomic analysis reveals insights into the response of Cryptolaemus montrouzieri to bottom-up transfer of cadmium and lead across a multi-trophic food chain

Contamination of agro-ecosystems with heavy metals can affect the development and reproduction of insect natural enemies. This study reports a detailed Tandem Mass Tag based quantitative proteomic analysis of underlying mechanisms responsible for stress response of Cryptolaemus montrouzieri against heavy metals (cadmium (Cd) and lead (Pb)) transported across a multi-trophic food chain. A total of 6639 proteins were detected under Cd as well as Pb stress. In Pb versus the control cluster, 69 proteins (28 up-regulated and 41 down-regulated) were differentially expressed whereas 268 proteins were differentially expressed under Cd versus the control cluster, having 198 proteins up-regulated and 70 down-regulated proteins. The analysis of differentially expressed proteins showed that 27 proteins overlapped in both clusters representing the core proteome to Pb and Cd stress. The bioinformatics analysis demonstrated that these proteins were mapped to 57 and 99 pathways in Pb versus control and Cd versus control clusters, respectively. The functional classification by COG, GO and KEGG databases showed significant changes in protein expression by C. montrouzieri under Pb and Cd stress. The heavy metal stress (Pb and Cd) induced significant changes in expression of proteins like hexokinase (HK), succinyl-CoA, trypsin like proteins, cysteine proteases, cell division cycle proteins, and yellow gene proteins. The results provide detailed information on the protein expression levels of C. montrouzieri and will serve as basic information for future proteomic studies on heavy metal responses of insect predators within a multi-trophic food chain.

Abstract 3923: Ubiquitin ligases implicated as predictive biomarkers for poor outcome to immunotherapy in melanoma patients

Abstract Background From a medical standpoint, melanoma is considered the most serious form of skin cancer. Immunotherapy treatment regimes frequently target the PD1 pathway and can be highly effective. Despite successes some patients remain unresponsive and a better understanding of what underlies these non-responders could enable better patient treatment stratification and an overall higher positive response rate. Here using unbiased mass spectrometry (LC-MS) proteomics profiling, we sought to identify protein biomarker signatures in patients with metastatic melanoma that correlate with the patients’ intervention responses. Via this discovery-based approach we identify a panel of protein biomarkers that correlate with an improved treatment response and could serve as a panel for patient stratification prior to commencement of immunotherapy regimes. Methods Formalin-fixed paraffin-embedded (FFPE) tumor samples were used for unbiased quantification of proteins using data-independent acquisition (DIA) LC-MS technology and Biognosys’ Spectronaut software. Patient samples were analyzed at the start of treatment (PD1 inhibitor) and classified as responders (n = 9) or non-responders (n = 15) at 3 months post-treatment. Significant protein expression changes between the two groups was determined by t-test (p &amp;lt; 0.01, log2 fold-change &amp;gt; 0.58) and further machine learning-assisted analysis was conducted. Additionally, patient’s tumor mutational burden (TMB) was analyzed. Results Analysis of the FFPE samples from anti-PD1-treated metastatic melanoma patients resulted in the identification and quantification of more than 9,000 proteins in total with an average of nearly 8,000 proteins per sample. Stratification of the patients into responders and non-responders led to the identification of 103 proteins that are significantly up- or down-regulated between the groups. Interestingly, proteins associated with ubiquitination pathways were significantly enriched among the identified regulated proteins with 7 proteins having significantly lower abundance in responders when compared to non-responders including various members of the ubiquitin ligase family. Further, a proteomic signature was identified that characterizes the responder and non-responder groups. Conclusions In this study we demonstrate the power of unbiased proteomic profiling for the characterization of melanoma patients and their responses to PD1-targeted immunotherapy. We identify a set of biomarkers associated with ubiquitination pathways that correlate with treatment outcome although these findings need to further be confirmed in orthogonal cohorts. Citation Format: Nigel Beaton, Tobias Treiber, Anna Juncker-Jensen, Xin-Xing Tan, Julia Martinez Gomez, Mitchell Levesque, Jakob Vowinkel. Ubiquitin ligases implicated as predictive biomarkers for poor outcome to immunotherapy in melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3923.

Abstract WP210: Eicosapentaenoic Acid (EPA) Decreases Cytokine Release And Inflammatory Proteins In Brain Vascular Endothelium During Inflammation

Background: Brain vascular endothelial cell (EC) dysfunction is characterized by release of pro-inflammatory mediators that contribute to atherosclerosis and ischemic stroke. Treatment with icosapent ethyl (IPE), the ethyl ester of the omega-3 fatty acid, eicosapentaenoic acid (EPA), reduced first and total ischemic strokes each by 36%, without increasing hemorrhagic stroke, in statin-treated patients with elevated cardiovascular risk (REDUCE-IT). We tested the effects of EPA on cytokine release and expression of inflammatory proteins from brain microvascular ECs during inflammation. Methods: Human brain microvascular endothelial cells (HBECs) were pretreated with the cytokine IL-6 at 12 ng/ml for 2 h before treatment with EPA (40 μM) for 24 h. Proteomic analysis was performed using LC/MS to capture relative expression levels. Only significant changes in protein expression between treatment groups &gt;1-fold were analyzed. Gene set enrichment analysis was also performed on all significantly regulated proteins to identify groups of proteins (pathways) that were affected. Levels of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor-α (TNF-α) were measured by immunochemistry (ELISA). Results: IL-6 exposure produced increased levels of sICAM-1 and TNF-α by 102% and 147% (p&lt;0.001), respectively, in brain ECs compared with vehicle. EPA treatment reduced release of sICAM-1 by 43% (p&lt;0.001) and TNF-α by 52% (p&lt;0.001) compared to IL-6 alone. EPA also downregulated 43 proteins involved in the “neutrophil degranulation” pathway in brain ECs (p-adjusted = 2.63 х 10 -12 ), including heat shock protein 90-alpha and beta by 1.1-fold each relative to IL-6 alone. Conclusions: In brain vascular ECs, EPA significantly reduced expression of pro-inflammatory mediators and proteins associated with neutrophil degranulation under inflammatory conditions. The ability of EPA to reverse brain EC dysfunction during inflammatory may contribute to reductions in stroke risk.

Increased Desmosine in the lens capsules is associated with augmented elastin turnover in Pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PXF) is an idiopathic disease with a high prevalence rate. The elastosis disorder is contributed by genetic and non-genetic factors. Elastin dysregulation associated with the disease mechanism is incompletely understood. This study evaluated the molecules of the elastogenesis machinery in PXF. Lens capsule and aqueous humor (aqH) samples (age/sex-matched) were collected from the eyes with PXF alone and PXF with glaucoma (PXF-G) undergoing Extra Capsular Cataract Extraction (ECCE) surgery. The Elastin turnover was assessed by estimating Desmosine levels in the lens capsules by HPLC analysis. Expression of elastogenesis genes [EMILIN1, CLU, FBN1, FN1, FBLN5, FBLN4 and LOXL1] were evaluated in the lens capsule by qPCR while the proteins were assessed in aqH by western blot analysis.The Desmosine content in the lens capsules were 3-fold and 6-fold elevated in PXF (P = 0.02) and PXF-G (P = 0.01) respectively compared to the cataract-alone, indicating increased elastin degradation. A significant increase in the transcript levels of the CLU, FBLN4, EMILIN1, FBLN5, FN1, FBN1, LOXL1 along with significant changes in protein expression of CLU, FBLN5, FBN1 and LOXL1 signified up-regulation of the elastogenesis machinery. The study provides direct evidence of augmented elastin degradation and turnover in the lens capsule of PXF marked by increased Desmosine content and the expression of proteins involved in mature elastin formation.

Sex-specific effect of prenatal alcohol exposure on N-methyl-D-aspartate receptor function in orbitofrontal cortex pyramidal neurons of mice.

Alcohol consumption during pregnancy can produce behavioral and cognitive deficits that persist into adulthood. These include impairments in executive functions, learning, planning, and cognitive flexibility. We have previously shown that moderate prenatal alcohol exposure (PAE) significantly impairs reversal learning, a measure of flexibility mediated across species by different brain areas that include the orbital frontal cortex (OFC). Reversal learning is likewise impaired by genetic or pharmacological inactivation of GluN2B subunit-containing N-methyl-D-aspartate receptors (NMDARs). In the current study, we tested the hypothesis that moderate PAE persistently alters the number and function of GluN2B subunit-containing NMDARs in OFC pyramidal neurons of adult mice. We used a rodent model of fetal alcohol spectrum disorders and left offspring undisturbed until adulthood. Using whole-cell, patch-clamp recordings, we assessed NMDAR function in slices from 90- to 100-day-old male and female PAE and control mice. Pharmacologically isolated NMDA receptor-mediated evoked excitatory postsynaptic currents (NMDA-eEPSCs) were recorded in the absence and presence of the GluN2B antagonist, Ro25-6981(1µM). In a subset of littermates, we evaluated the level of GluN2B protein expression in the synaptic fraction using Western blotting technique. Our results indicate that PAE females show significantly larger (~23%) NMDA-eEPSC amplitudes than controls, while PAE induced a significant decrease (~17%) in NMDA-eEPSC current density of pyramidal neurons recorded in slices from male mice. NMDA-eEPSC decay time was not affected in PAE-exposed mice from either sex. The contribution of GluN2B subunit-containing NMDARs to the eEPSCs was not significantly altered by PAE. Moreover, there were no significant changes in protein expression in the synaptic fraction of either PAE males or females. These findings suggest that low-to-moderate PAE modulates NMDAR function in pyramidal neurons in a sex-specific manner, although we did not find evidence that the effect is mediated by dysfunction of synaptic GluN2B subunit-containing NMDARs.

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients.

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.

Renal Transporter Expression Changes in Rodent Models of Nonalcoholic Steatohepatitis

Inter-individual alterations to renal elimination processes can lead to adverse drug reactions. Nonalcoholic steatohepatitis (NASH) is known to alter hepatic drug transport but may also affect renal transporters as well. This study investigates renal physiological changes in rodent models of NASH for identification of a model that recapitulates human alterations. Rats on methionine and choline deficient (MCD), atherogenic (Athero) or control; Leprdb/db mice on MCD (db/db); C57BL/6J mice on fast food with thioacetamide (FFDTH), American lifestyle induced obesity syndrome (ALIOS) or control diet were used to characterize hepatic and renal pathology, clinical chemistry, and the transcriptional and translational expression of drug transporters by RT-qPCR and surrogate peptide LC-MS/MS, respectively. Hepatic pathology confirms development of NASH while renal pathology demonstrates no lesions except mild lipid accumulation in ALIOS. MCD, Athero and ALIOS exhibit an increase in blood urea nitrogen while all models, except Athero, demonstrate a decrease in GFR. There is a significant decrease in protein expression for renal basolateral uptake transporters, OCT3 and OATP4C1, for mouse models (from 1.97, 0.67 to 1.17, 0.35 db/db; 1.43, 0.27 FFDTH; 1.50, 0.34 ALIOS pmol/mg protein, respectively) but neither rat model. Similarly, renal apical uptake transporters, OAT5 and OATP1A1, exhibit a significant decrease in mouse models (from 4.59, 0.69 to 0.45, 0.02 db/db; 1.59, 0.21 FFDTH; 2.83, 0.36 ALIOS pmol/mg protein, respectively) but a significant increase of OAT5 in MCD (1.67 to 4.17 pmol/mg protein). Renal apical efflux transporters elicit a variety of significant changes in protein expression including an increase of MRP2 in MCD (0.17 to 0.30 pmol/mg protein), decrease of BCRP in db/db and ALIOS (from 0.25 to 0.14, 0.13 pmol/mg protein, respectively) and a decrease of MRP4 in mouse models (from 0.87 to 0.34 db/db; 0.29 FFDTH; 0.18 ALIOS pmol/mg protein). The only significant change in renal basolateral efflux transporters is a decrease in MRP3 protein in MCD and Athero (from 2.71 to 1.53, 1.68 pmol/mg protein, respectively). These data suggest variations in renal physiology are elicited by NASH in a range of rodent models. This demonstrates the potential variability in renal elimination of drugs during NASH and provides a resource to identify the appropriate model for future studies.

MicroRNA-3148 acts as molecular switch promoting malignant transformation and adipocytic differentiation of immortalized human bone marrow stromal cells via direct targeting of the SMAD2/TGF\u03b2 pathway

MicroRNAs (miRs/miRNAs) play a key role in posttranscriptional regulation of gene expression and are implicated in a number of physiological and pathological conditions, including cellular malignant transformation. In the current study, we investigated the role of miR-3148 in regulating human stromal (mesenchymal) stem cell (hMSC) differentiation and transformation. Stable expression of miR-3148 in telomerized hMSC (hMSC-miR-3148) led to significant increase in in vitro adipocytic differentiation and suppression of osteoblastic differentiation. Concordantly, global gene expression profiling revealed significant enrichment in cholesterol biosynthesis pathway, and pathways related to enhanced cell movement and survival, whereas processes related to bone and connective tissue developments, cell death, apoptosis, and necrosis were downregulated. Global proteomic analysis using 2D-DIGE followed by mass spectrometry (MS) revealed significant changes in protein expression in hMSC-miR-3148 and enrichment in protein networks associated with carcinogenesis. Functional studies revealed that hMSC-miR-3148 exhibited enhanced in vitro cell proliferation, colony formation, migration, invasion, sphere formation, doxorubicin resistance, and increased active number of cells in S and G2/M cell cycle phases and formed sarcoma-like tumors with adipocyte infiltration when implanted into immunocompromised mice. SMAD2 was identified as bone fide gene target for miR-3148 using qRT-PCR, Western blotting, and UTR-based reporter assay. In agreement with our data, SMAD2 expression was downregulated in 47% of patients with soft tissue sarcoma. Bioinformatics analysis revealed that elevated miR-3148 expression correlates with poor prognosis in several human cancer types, including sarcoma. Our study identified miR-3148 as factor regulating hMSC differentiation and is involved in promoting malignant transformation of telomerized hMSC.

Quantitative proteomics analysis of high and low polyphenol expressing recombinant inbred lines (RILs) of peanut (Arachis hypogaea L.)

To facilitate selective breeding of polyphenol-rich peanuts, we looked for mass spectrometry-based proteomic evidence, investigating a subset of recombinant inbred lines (RILs) developed by the Australian peanut breeding program. To do this, we used label-free shotgun proteomics for protein and peptide quantitation, statistically analyzed normalized spectral abundance factors using R-package, as well as assayed important antioxidants. Results revealed statistically significant protein expression changes in 82 proteins classified between high or low polyphenols expressing RILs. Metabolic changes in polyphenol-rich RIL p27-362 point towards increased enzymatic breakdown of sugars and phenylalanine biosynthesis. The study revealed phenylpropanoid pathway overexpression resulting in increased polyphenols biosynthesis. Overexpression of antioxidant enzymes such as catalase, by 73.4 fold was also observed. A strong metabolic correlation exists with the observed phenotypic traits. Peanut RIL p27-362 presents a superior nutritional composition with antioxidant-rich peanut phenotype and could yield commercial profits. Data are available via ProteomeXchange with identifierPXD015493.

Quinolinate as a Marker for Kynurenine Metabolite Formation and the Unresolved Question of NAD+ Synthesis During Inflammation and Infection.

Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite in the kynurenine pathway of tryptophan catabolism leading to the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As a precursor for NAD+, Quin can direct a portion of tryptophan catabolism toward replenishing cellular NAD+ levels in response to inflammation and infection. Intracellular Quin levels increase dramatically in response to immune stimulation [e.g., lipopolysaccharide (LPS) or pokeweed mitogen (PWM)] in macrophages, microglia, dendritic cells, and other cells of the immune system. NAD+ serves numerous functions including energy production, the poly ADP ribose polymerization (PARP) reaction involved in DNA repair, and the activity of various enzymes such as the NAD+-dependent deacetylases known as sirtuins. We used highly specific antibodies to protein-coupled Quin to delineate cells that accumulate Quin as a key aspect of the response to immune stimulation and infection. Here, we describe Quin staining in the brain, spleen, and liver after LPS administration to the brain or systemic PWM administration. Quin expression was strong in immune cells in the periphery after both treatments, whereas very limited Quin expression was observed in the brain even after direct LPS injection. Immunoreactive cells exhibited diverse morphology ranging from foam cells to cells with membrane extensions related to cell motility. We also examined protein expression changes in the spleen after kynurenine administration. Acute (8 h) and prolonged (48 h) kynurenine administration led to significant changes in protein expression in the spleen, including multiple changes involved with cytoskeletal rearrangements associated with cell motility. Kynurenine administration resulted in several expression level changes in proteins associated with heat shock protein 90 (HSP90), a chaperone for the aryl-hydrocarbon receptor (AHR), which is the primary kynurenine metabolite receptor. We propose that cells with high levels of Quin are those that are currently releasing kynurenine pathway metabolites as well as accumulating Quin for sustained NAD+ synthesis from tryptophan. Further, we propose that the kynurenine pathway may be linked to the regulation of cell motility in immune and cancer cells.

Abstract P3-11-15: A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance is due to upregulation of growth factor pathways (i.e., HER2 and EGFR) and increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells (LTLT-Ca). However, many postmenopausal women with AI-resistance tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor (ER+) positive and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer using a combination approach of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation and motility. To test this hypothesis, T47Darom cells (T47D cells which stably overexpress aromatase) and T47DaromLR cells (T47Darom cells that are letrozole resistant) were treated with lapatinib, glyceollin, or the combination. To evaluate viability, resazurin assays were performed and results demonstrated that glyceollin and/or lapatinib had no effect on proliferation in the T47Darom cells. However, glyceollin alone and glyceollin + lapatinib inhibited proliferation of T47DaromLR cells by 46% and 59%, respectively. Cell survival was assessed by colony formation assays and glyceollin caused a 33% reduction in cell survival and glyceollin + lapatinib caused a 60% reduction in the colony formation of T47Darom cells. Interestingly, in T47DaromLR cells glyceollin and the combination significantly inhibited cell survival by 94% alone and 96%. To further evaluate changes that occur as AI sensitive cells transition to AI resistance, a quantitative proteomic analysis of the whole cell lysates of T47DaromLR (AI-resistant) versus T47Darom cells (AI-sensitive) was performed to identify significant protein expression changes. Results demonstrated a 3.195-fold-increase (p&amp;lt;0.01) in protein disulfide isomerase (P4HB) and a 0.484-fold decrease (p&amp;lt;0.01) in peptidyl-prolyl cis-trans isomerase (FKBP4) which was validated by immunoblotting. While the molecular mechanism by which P4HB contributes to tumorigenesis and metastasis is unknown, it has been demonstrated to be involved in the proliferation, survival, and metastasis of several types of cancer cells. FKBP4 is also of interest as it has been shown to be involved in mitotic arrest and apoptotic signaling. To interrogate the effect of combination therapy on HER effector signaling cascades, western blot analyses were performed to establish the endogenous expression of various proteins. Results demonstrated ERα expression was increased while HER2 was decreased in T47Darom cells compared to T47DaromLR cells. The protein expression findings from the T47D variant cell lines followed a similar trend as those previously reported in LTLT-Ca cells and their AI-sensitive counterparts. Taken together, these results suggest that dual inhibition using glyceollins and lapatinib may have potential as a novel combination therapy approach for postmenopausal patients with hormone-dependent, letrozole-resistant breast tumors. This work was also supported by NIH grant 1SC1GM126617 awarded to SL Tilghman. Citation Format: Rashidra R Walker, Jankiben Patel, A. Michael Davidson, Karen Gallegos, Syreeta L Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-15.

Effects of spaceflight on the mouse submandibular gland

ObjectiveThis study was conducted to determine if the morphology and biochemistry of the mouse submandibular gland is affected by microgravity and the spaceflight environment. DesignTissues from female mice flown on the US space shuttle missions Space Transportation System (STS)-131 and STS-135 for 15 and 13 d, respectively, and from male mice flown on the 30 d Russian Bion-M1 biosatellite, were examined using transmission electron microscopy and light and electron microscopic immunohistochemistry. ResultsIn contrast to the parotid gland, morphologic changes were not apparent in the submandibular gland. No significant changes in protein expression, as assessed by quantitative immunogold labeling, occurred in female mice flown for 13–15 d. In male mice, however, increased labeling for salivary androgen binding protein alpha (in acinar cell secretory granules), and epidermal growth factor and nerve growth factor (in granular convoluted duct cell granules) was seen after 30 d in space. ConclusionThese results indicate that spaceflight alters secretory protein expression in the submandibular gland and suggest that the sex of the animals and the length of the flight may affect the response. These findings also show that individual salivary glands respond differently to spaceflight. Saliva contains proteins secreted from salivary glands and is easily collected, therefore is a useful biofluid for general medical analyses and in particular for monitoring the physiology and health of astronauts.

Proteomic Profile of a Chronic Binge Ethanol Exposure Model.

Chronic binge alcohol drinking is known to increase risky decision through pathological impulsive behaviors. Recently, we established a novel rodent model of ethanol-induced waiting impulsivity using 5-choice serial reaction time task (5-CSRTT) in mice. However, molecular mechanisms underlying the chronic binge ethanol-induced waiting impulsivity is not well characterized. Among brain regions involved in impulsivity, the anterior cingulate cortex (ACC) is a major neural substrate for mediating the 5-CSRTT-based waiting impulsivity. Thus, we sought to determine the ACC proteomic profile using label-free proteomics of mice exhibiting ethanol-induced impulsivity. Ingenuity pathway analysis revealed that impulsivity-related proteins involved in ion channel complexes such as KCNIP3 (potassium voltage-gated channel interacting protein 3) and CACNG2 (calcium voltage-gated channel auxiliary subunit gamma 2) are downregulated in the ACC. We identified significant protein expression changes in the mechanistic target of rapamycin (mTOR) canonical pathway between control and ethanol-induced impulsive mice. Impulsive mice showed over 60% of proteins involved in the mTOR canonical pathway have been altered. This pathway has been previously implicated in the neuroadaptation in drugs of abuse and impulsivity. We found substantial changes in the protein levels involved in neurological disorders such as schizophrenia and Alzheimer's disease. Our findings provide a neuroproteomic profile of ethanol-induced impulsive mice.

Proteomic Analysis of Mouse Brain Subjected to Spaceflight

There is evidence that spaceflight poses acute and late risks to the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched from the Kennedy Space Center (KSC) on a 13-day mission. Within 3–5 h after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the gray and/or white matter. While there was no overlap between the white and gray matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.