Analysis Of Proteomic Changes Research Articles

Loss-of-function of RNA-binding protein PRRC2B causes translational defects and congenital cardiovascular malformation.

Alternative splicing generates variant forms of proteins for a given gene and accounts for functional redundancy or diversification. A novel RNA-binding protein, Pro-rich Coiled-coil Containing Protein 2B (PRRC2B), has been reported by multiple laboratories to mediate uORF-dependent and independent regulation of translation initiation required for cell cycle progression and proliferation. We identified two alternative spliced isoforms in human and mouse hearts and HEK293T cells, full-length (FL) and exon 16-excluded isoform ΔE16. A congenital heart disease-associated human mutation-mimicry knock-in of the equivalent variant in the mouse genome leads to the depletion of the full-length Prrc2b mRNA but not the alternative spliced truncated form ΔE16, does not cause any apparent structural or functional disorders. In contrast, global genetic inactivation of the PRRC2B gene in the mouse genome, nullifying both mRNA isoforms, caused patent ductus arteriosus (PDA) and neonatal lethality in mice. Bulk and single nucleus transcriptome profiling analyses of embryonic mouse hearts demonstrated a significant overall downregulation of multiple smooth muscle-specific genes in Prrc2b mutant mice resulting from reduced smooth muscle cell number. Integrated analysis of proteomic changes in Prrc2b null mouse embryonic hearts and polysome-seq and RNA-seq multi-omics analysis in human HEK293T cells uncover conserved PRRC2B-regulated target mRNAs that encode essential factors required for cardiac and vascular development. Our findings reveal the connection between alternative splicing regulation of PRRC2B, PRRC2B-mediated translational control, and congenital cardiovascular development and disorder. This study may shed light on the significance of PRRC2B in human cardiovascular disease diagnosis and treatment.

Abstract A018: Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells

Abstract Introduction: Enzalutamide is indicated for treatment of castrate-resistant prostate cancer (CRPC), but therapeutic effect is transient, with improvement in survival of approximately 4 months. The mechanisms by which prostate cancer (PCa) cells survive Enzalutamide therapy are poorly understood, but androgen receptor (AR) and non-AR mediated mechanisms have been reported from transcriptome studies. We performed unbiased high resolution proteomic analyses to identify proteins and pathways that are involved in survival and progression in Enzalutamide treatment failure. Materials & Methods: Enzalutamide-resistant MR49F and MR42D cell lines were maintained in standard medium (DMEM with 5% FBS) with Enzalutamide 10µM. Parental LNCaP cells were grown in standard medium. For perturbation, MR49F and MR42D cells were then changed to standard medium with either Enzalutamide 10µM (ENZA) or DMSO (control) for 7 days in. Medium was changed every 48 hours. Protein lysates from 4 biological replicates were labeled using a Tandem Mass Tag (TMT) assay and subjected to mass spectrometry (MS). Protein abundance was measured and significant differences in expression were analysed using a 2-way t-test with a permutation based false detection rate (FDR) of 0.05 and 250 randomisations in the Perseus software suite. MR49F and MR42D were compared to parental LNCaP, and MR49F and MR42D were compared on and off Enzalutamide treatment pressure. Functional biological pathways involved were identified using the DAVID and WebGestalt online pathway analysis tools. Results: MS identified 5364 proteins. Of these, 247 (180 up, 67 down) were differentially expressed by at least 50% in both MR49F and MR42D cell lines relative to parental LNCaP cells. When MR49F and MR42D cell lines were grown with or without Enzalutamide pressure, 5 proteins (4 up, 1 down) were regulated by at least 50% in both cell lines (ANLN, NIN, UQCRB, HIST1H3A and S100P). Pathway analyses with the differentially expressed and regulated proteins identified perturbations in biological pathways with a high representation of pathways related to metabolism, including amino acid and fatty acid metabolism, ascorbate and propanoate metabolism, drug transport and metabolism, and cell adhesion molecules. Conclusions: This unbiased proteomic analysis of Enzalutamide-resistant PCa cells identified proteins and pathways that may be essential for PCa cell survival and progression during Enzalutamide therapy failure. Enzalutamide therapy failure appears to involve some non-AR mediated mechanisms which may be targeted for therapeutic benefit. A better understanding of the mechanisms of Enzalutamide resistance could lead to development of novel therapies for post-Enzalutamide disease progression. Citation Format: Chidi N. Molokwu, Anders Kristensen, Chris Sutton. Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A018.

Cell-Type-Specific Signalling Networks Impacted by Prostate Epithelial-Stromal Intercellular Communication.

Prostate cancer is the second most common cause of cancer death in males. A greater understanding of cell signalling events that occur within the prostate cancer tumour microenvironment (TME), for example, between cancer-associated fibroblasts (CAFs) and prostate epithelial or cancer cells, may identify novel biomarkers and more effective therapeutic strategies for this disease. To address this, we used cell-type-specific labelling with amino acid precursors (CTAP) to define cell-type-specific (phospho)proteomic changes that occur when prostate epithelial cells are co-cultured with normal patient-derived prostate fibroblasts (NPFs) versus matched CAFs. We report significant differences in the response of BPH-1 benign prostate epithelial cells to CAF versus NPF co-culture. Pathway analysis of proteomic changes identified significant upregulation of focal adhesion and cytoskeleton networks, and downregulation of metabolism pathways, in BPH-1 cells cultured with CAFs. In addition, co-cultured CAFs exhibited alterations in stress, DNA damage, and cytoskeletal networks. Functional validation of one of the top differentially-regulated proteins in BPH-1 cells upon CAF co-culture, transglutaminase-2 (TGM2), demonstrated that knockdown of this protein significantly reduced the proliferation and migration of prostate epithelial cells. Overall, this study provides novel insights into intercellular communication in the prostate cancer TME that may be exploited to improve patient management.

Comparative Analysis of the Response to Polyethylene Glycol-Simulated Drought Stress in Roots from Seedlings of "Modern" and "Ancient" Wheat Varieties.

Durum wheat is widely cultivated in the Mediterranean, where it is the basis for the production of high added-value food derivatives such as pasta. In the next few years, the detrimental effects of global climate change will represent a serious challenge to crop yields. For durum wheat, the threat of climate change is worsened by the fact that cultivation relies on a few genetically uniform, elite varieties, better suited to intensive cultivation than "traditional" ones but less resistant to environmental stress. Hence, the renewed interest in "ancient" traditional varieties are expected to be more tolerant to environmental stress as a source of genetic resources to be exploited for the selection of useful agronomic traits such as drought tolerance. The aim of this study was to perform a comparative analysis of the effect and response of roots from the seedlings of two durum wheat cultivars: Svevo, a widely cultivated elite variety, and Saragolla, a traditional variety appreciated for its organoleptic characteristics, to Polyethylene glycol-simulated drought stress. The effect of water stress on root growth was analyzed and related to biochemical data such as hydrogen peroxide production, electrolyte leakage, membrane lipid peroxidation, proline synthesis, as well as to molecular data such as qRT-PCR analysis of drought responsive genes and proteomic analysis of changes in the protein repertoire of roots from the two cultivars.

Analysis of proteomic changes in cassava cv. Kasetsart 50 caused by Sri Lankan cassava mosaic virus infection.

Sri Lankan cassava mosaic virus (SLCMV) is a plant virus causing significant economic losses throughout Southeast Asia. While proteomics has the potential to identify molecular markers that could assist the breeding of virus resistant cultivars, the effects of SLCMV infection in cassava have not been previously explored in detail. Liquid Chromatography-Tandem Mass Spectrometry (LC/MS-MS) was used to identify differentially expressed proteins in SLCMV infected leaves, and qPCR was used to confirm changes at mRNA levels. LC/MS-MS identified 1,813 proteins, including 479 and 408 proteins that were upregulated in SLCMV-infected and healthy cassava plants respectively, while 109 proteins were detected in both samples. Most of the identified proteins were involved in biosynthetic processes (29.8%), cellular processes (20.9%), and metabolism (18.4%). Transport proteins, stress response molecules, and proteins involved in signal transduction, plant defense responses, photosynthesis, and cellular respiration, although present, only represented a relatively small subset of the detected differences. RT-qPCR confirmed the upregulation of WRKY 77 (A0A140H8T1), WRKY 83 (A0A140H8T7), NAC 6 (A0A0M4G3M4), NAC 35 (A0A0M5JAB4), NAC 22 (A0A0M5J8Q6), NAC 54 (A0A0M4FSG8), NAC 70 (A0A0M4FEU9), MYB (A0A2C9VER9 and A0A2C9VME6), bHLH (A0A2C9UNL9 and A0A2C9WBZ1) transcription factors. Additional upregulated transcripts included receptors, such as receptor-like serine/threonine-protein kinase (RSTK) (A0A2C9UPE4), Toll/interleukin-1 receptor (TIR) (A0A2C9V5Q3), leucine rich repeat N-terminal domain (LRRNT_2) (A0A2C9VHG8), and cupin (A0A199UBY6). These molecules participate in innate immunity, plant defense mechanisms, and responses to biotic stress and to phytohormones. We detected 1,813 differentially expressed proteins infected cassava plants, of which 479 were selectively upregulated. These could be classified into three main biological functional groups, with roles in gene regulation, plant defense mechanisms, and stress responses. These results will help identify key proteins affected by SLCMV infection in cassava plants.

Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli.

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations. Data is available via ProteomeXchange with identifier PXD026884.

Comprehensive chemical proteomics analyses reveal that the new TRi-1 and TRi-2 compounds are more specific thioredoxin reductase 1 inhibitors than auranofin

Anticancer drugs that target cellular antioxidant systems have recently attracted much attention. Auranofin (AF) is currently evaluated in several clinical trials as an anticancer agent that targets the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase, TXNRD1 and TXNRD2. Recently, two novel TXNRD1 inhibitors (TRi-1 and TRi-2) have been developed that showed anticancer efficacy comparable to AF, but with lower mitochondrial toxicity. However, the cellular action mechanisms of these drugs have not yet been thoroughly studied. Here we used several proteomics approaches to determine the effects of AF, TRi-1 and TRi-2 when used at IC50 concentrations with the mouse B16 melanoma and LLC lung adenocarcinoma cells, as these are often used for preclinical mouse models in evaluation of anticancer drugs. The results demonstrate that TRi-1 and TRi-2 are more specific TXNRD1 inhibitors than AF and reveal additional AF-specific effects on the cellular proteome. Interestingly, AF triggered stronger Nrf2-driven antioxidant responses than the other two compounds. Furthermore, AF affected several additional proteins, including GSK3A, GSK3B, MCMBP and EEFSEC, implicating additional effects on glycogen metabolism, cellular differentiation, inflammatory pathways, DNA replication and selenoprotein synthesis processes. Our proteomics data provide a resource for researchers interested in the multidimensional analysis of proteome changes associated with oxidative stress in general, and the effects of TXNRD1 inhibitors and AF protein targets in particular.

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly.

ABSTRACTCiliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans, the impairment of these motors through mutation results in the disease primary ciliary dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less-known assembly factor DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, The only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

Drug Screening Reveals That Ibrutinib (PCI-32765) Exhibits Synergy With BCL-2 and Proteasome Inhibitors In Models Of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL)

BackgroundIbrutinib, a Bruton's Tyrosine Kinase inhibitor, is clinically effective in relapsed MCL and CLL, but responses are often partial and resistance eventually develops. To identify agents that synergize with ibrutinib, we performed an in vitro combinatorial drug screen and evaluated the most promising combinations in additional models. MethodsDrug screens were performed in JVM2 and Z138 MCL cell lines, combining ibrutinib with a diverse panel of secondary agents inhibiting targets within the B-cell receptor (BCR) pathway (SC514, PD325901, enzastaurin, R788, temsirolimus, dasatinib, idelalisib) and agents inhibiting targets outside the BCR pathway (ABT-199, bendamustine, carfilzomib, bortezomib, geldanamycin, panobinostat, PD0332991). Three doses (IC10, IC20, IC 30) for each drug were chosen and combined in a 3x3 format. Bliss Model of Additivity was used to quantitate synergistic cytotoxicity. Levels of cleaved PARP or Annexin V/PI staining were assessed for cell lines exposed to ibrutinib and synergistic agents, including bortezomib, carfilzomib, and ABT-199. The Ibrutinib + carfilzomib combination was further tested in MCL cell lines Rec1 and Jeko, in primary MCL and CLL patient (pt) samples, and in a MCL-SCID-hu mouse. Molecular analysis of the mechanistic interactions of the agents has been initiated using forward-phase antibody arrays (R and D Systems). ResultsInhibitors of targets within the BCR pathway were ineffective at potentiating the effects of ibrutinib (magnitude of synergy ranging -12%-9% in Z138, -25%-8% in JVM2). However, substantial synergistic cytotoxicity was seen with some agents that target outside the BCR pathway (magnitude of synergy ranging -5%-37% in Z138, -17%-48% in JVM2). Notably, we observed robust and reproducible synergistic cytotoxicity by combining ibrutinib with proteasome inhibitors carfilzomib and bortezomib as well as BCL-2 inhibitor ABT-199.The degree of synergyof ibrutinib + ABT-199 in MCL cell lines was durable across all 9 dosing combinations, (23-32% in Z-138, 10-26% in JVM-2). In CLL pt samples, ibrutinib + ABT-199 conferred modest synergy (10-20%) and near-complete cytotoxicity (61-70%). Flow cytometry for cleaved PARP demonstrated a high degree of apoptosis when combining ibrutinib with ABT-199 (94.5% in Z-138, 82.1% in Z-138).At optimal dosing, bortezomib conferred 19% synergy with ibrutinib in Z138 and 27% in JVM2. The best synergistic effect of carfilzomib and ibrutinib was 50% in Z138 and 65% in JVM2. The synergistic effect between carfilzomib and ibrutinib was maintained in Rec1 and Jeko MCL cell lines and in primary MCL pt samples. In CLL pt samples, ibrutinib + proteasome inhibition also conferred high degrees of total cytotoxicity (58-71% for carfilzomib, 42-62% for bortezomib). Flow cytometry for cleaved PARP or Annexin V/PI demonstrated increased apoptosis for ibrutinib +carfilzomib (92.3% in Z138, 61.2% in JVM2, 95% in Rec1, 90% in Jeko). The combination treatment increased the percent survival of MCL-SCID-hu mice in three-fold over single agent treatment in a 125-day trial.Preliminary analysis of proteomic changes induced by ibrutinib+ carfilzomib indicates synergistic alterations in cell cycle proteins and in apoptosis-regulating proteins, although the specific alterations differ between Z138 and JVM2 cell lines. DiscussionWhile agents within the BCR pathway did not display synergistic cytotoxicity with ibrutinib in our drug screen, several agents outside the BCR pathway did synergize with ibrutinib, notably ABT-199, bortezomib, and carfilzomib in two MCL cell lines. For all three drug combinations, this effect appeared to be mediated via apoptosis. The synergistic effect of ibrutinib + carfilzomib was further studied in 2 additional MCL cell lines, 3 MCL pt samples, and CLL pt samples. MCL-SCID-hu mice demonstrated three-fold increased survival when treated in combination over animals treated with either agent alone. The identification of unexpected synergistic cytotoxic interactions between inhibitors of apparently unrelated pathways is a benefit of our combinatorial screening approach, although the molecular mechanisms remain to be elucidated. The combination of ibrutinib with either proteasome or BCL-2 inhibition is proposed as promising novel treatments for MCL and CLL. A phase I/II clinical trial has been designed to test ibrutinib + carfilzomib in relapsed or refractory MCL and CLL. Disclosures:Williams:Celgene: Consultancy, Independent Data Safety Committee member Other, Research Funding; Genentech: Consultancy, Research Funding; Janssen: Research Funding; Millennium: Consultancy, Independent Data Safety Committee member, Independent Data Safety Committee member Other, Research Funding; Pharmacyclics: Consultancy, Research Funding; Onyx: Research Funding; Gilead: Research Funding. Wang:Onyx: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Novartis: Research Funding.

DIA proteomics analysis through serum profiles reveals the significant proteins as candidate biomarkers in women with PCOS

BackgroundThe aim of this study was to apply proteomic methodology for the analysis of proteome changes in women with polycystic ovary syndrome (PCOS).Material and methodsAll the participators including 31 PCOS patients and 31 healthy female as controls were recruited, the clinical characteristics data was recorded at the time of recruitment, the laboratory biochemical data was detected. Then, a data-independent acquisition (DIA)-based proteomics method was performed to compare the serum protein changes between PCOS patients and controls. In addition, Western blotting was used to validate the expression of identified proteomic biomarkers.ResultsThere were 80 proteins differentially expressed between PCOS patients and controls significantly, including 54 downregulated and 26 upregulated proteins. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that downregulated proteins were enriched in platelet degranulation, cell adhesion, cell activation, blood coagulation, hemostasis, defense response and inflammatory response terms; upregulated proteins were enriched in cofactor catabolic process, hydrogen peroxide catabolic process, antioxidant activity, cellular oxidant detoxification, cellular detoxification, antibiotic catabolic process and hydrogen peroxide metabolic process. Receiver operating characteristic curves analysis showed that the area under curve of Histone H4 (H4), Histone H2A (H2A), Trem-like transcript 1 protein (TLT-1) were all over than 0.9, indicated promising diagnosis values of these proteins. Western blotting results proved that the detected significant proteins, including H4, H2A, TLT-1, Peroxiredoxin-1, Band 3 anion transport protein were all differently expressed in PCOS and control groups significantly.ConclusionThese proteomic biomarkers provided the potentiality to help us understand PCOS better, but future studies comparing systemic expression and exact role of these candidate biomarkers in PCOS are essential for confirmation of this hypothesis.

Serpin neuropathology in the P497S UBQLN2 mouse model of ALS/FTD.

Accumulating evidence suggests X‐linked dominant mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD) through both loss‐ and gain‐of‐function mechanisms. However, the mechanisms by which the mutations cause disease are still unclear. The goal of the study was to uncover the possible pathomechanism(s) by which UBQLN2 mutations cause ALS/FTD. An analysis of proteomic changes in neuronal tissue was used to identify proteins with altered accumulation in the P497S UBQLN2 transgenic mouse model of ALS/FTD. We then used immunocytochemistry and biochemical techniques to confirm protein changes in the mutant P497S mice. Additionally, we used cell lines inactivated of UBQLN2 expression to determine whether its loss underlies the alteration in the proteins seen in P497S mice. The proteome screen identified a dramatic alteration of serine protease inhibitor (serpin) proteins in the mutant P497S animals. Double immunofluorescent staining of brain and spinal cord tissues of the mutant and control mice revealed an age‐dependent change in accumulation of Serpin A1, C1, and I1 in puncta whose staining colocalized with UBQLN2 puncta in the mutant P497S mice. Serpin A1 aggregation in P497S animals was confirmed by biochemical extraction and filter retardation assays. A similar phenomenon of serpin protein aggregation was found in HeLa and NSC34 motor neuron cells with inactivated UBQLN2 expression. We found aberrant aggregation of serpin proteins, particularly Serpin A1, in the brain and spinal cord of the P497S UBQLN2 mouse model of ALS/FTD. Similar aggregation of serpin proteins was found in UBQLN2 knockout cells suggesting that serpin aggregation in the mutant P497S animals may stem from loss of UBQLN2 function. Because serpin aggregation is known to cause disease through both loss‐ and gain‐of‐function mechanisms, we speculate that their accumulation in the P497S mouse model of ALS/FTD may contribute to disease pathogenesis through similar mechanism(s).

Exposure of Agaricus bisporus to Trichoderma aggressivum f. europaeum leads to growth inhibition and induction of an oxidative stress response

Green mould disease of mushroom, Agaricus bisporus,is caused by Trichodermaspecies and can result in substantial crop losses.Label free proteomic analysis of changes in the abundance of A. bisporusproteins following exposure to T. aggressivumsupernatantin vitroindicated increased abundance of proteins associated with an oxidative stress response (zinc ion binding (+6.6 fold); peroxidase activity (5.3-fold); carboxylic ester hydrolase (+2.4 fold); dipeptidase (+3.2 fold); [2Fe-2S] cluster assembly (+3.3 fold)). Proteins that decreased in relative abundance were associated with growth: structural constituent of ribosome, translation (-12 fold), deadenylation-dependent decapping of nuclear-transcribed mRNA (-3.4 fold), and small GTPase mediated signal transduction (-2.6 fold). In vivoanalysis revealed that 10-4 T. aggressivuminoculum decreased the mushroom yield by 29% to 56% and 10-3 T. aggressivuminoculum decreased the mushroom yield by 68% to 100%. Proteins that increased in abundance in A. bisporusin vivofollowing exposure to T. aggressivumindicated an oxidative stress response and included proteins with pyruvate kinase activity (+2.6 fold) and hydrolase activity (+2.1 fold)). The results indicate that exposure of A. bisporusmycelium to T. aggressivum in vitroand in vivoresulted in an oxidative stress response and reduction in growth.

Involvement of Cholesterol Metabolic Pathways in Recovery from Noise-Induced Hearing Loss.

The objective of this study was to explore the molecular mechanisms of acute noise-induced hearing loss and recovery of steady-state noise-induced hearing loss using miniature pigs. We used miniature pigs exposed to white noise at 120 dB (A) as a model. Auditory brainstem response (ABR) measurements were made before noise exposure, 1 day and 7 days after noise exposure. Proteomic Isobaric Tags for Relative and Absolute Quantification (iTRAQ) was used to observe changes in proteins of the miniature pig inner ear following noise exposure. Western blot and immunofluorescence were performed for further quantitative and qualitative analysis of proteomic changes. The average ABR-click threshold of miniature pigs before noise exposure, 1 day and 7 days after noise exposure, were 39.4 dB SPL, 67.1 dB SPL, and 50.8 dB SPL, respectively. In total, 2,158 proteins were identified using iTRAQ. Both gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses showed that immune and metabolic pathways were prominently involved during the impairment stage of acute hearing loss. During the recovery stage of acute hearing loss, most differentially expressed proteins were related to cholesterol metabolism. Western blot and immunofluorescence showed accumulation of reactive oxygen species and nuclear translocation of NF-κB (p65) in the hair cells of miniature pig inner ears during the acute hearing loss stage after noise exposure. Nuclear translocation of NF-κB (p65) may be associated with overexpression of downstream inflammatory factors. Apolipoprotein (Apo) A1 and Apo E were significantly upregulated during the recovery stage of hearing loss and may be related to activation of cholesterol metabolic pathways. This is the first study to use proteomics analysis to analyze the molecular mechanisms of acute noise-induced hearing loss and its recovery in a large animal model (miniature pigs). Our results showed that activation of metabolic, inflammatory, and innate immunity pathways may be involved in acute noise-induced hearing loss, while cholesterol metabolic pathways may play an important role in recovery of hearing ability following noise-induced hearing loss.

Characterisation of the interaction of Pseudomonas putida and Pseudomonas tolaasii with Trichoderma aggressivum

Green mould disease is caused by Trichoderma aggressivum which colonizes mushroom compost and reduces yield. Two Pseudomonas species are associated with mushroom compost: Pseudomonas putida, which stimulates mushroom pinning, and Pseudomonas tolaasii which has a negative effect on crop production. The aim of this work was to characterize T. aggressivum – Pseudomonas interactions as these may be important factors in the development of green mould disease. P. tolaasii supernatant inhibited growth by 57% but P. putida stimulated growth of T.aggressivum by 44%. Tolaasin production was identified in P. tolaasii cultures with a peak at 96 h. Fluorescent microscopy examination of T. aggressivum hyphae revealed that exposure to P. tolaasii supernatant decreased mycelial formation while increasing the abundance of conidia. Label free proteomic analysis of changes in the abundance of T. aggressivum proteins indicated that exposure to P. tolaasii supernatant lead to an oxidative stress response and catabolic enzyme activation (mitochondrial import inner membrane translocase complex (5.7-fold), oxidoreductase (5.2-fold), glucoamylase (5.1-fold)). Exposure of T. aggressivum to P. putida supernatant lead to an increase in the abundance of proteins associated with growth and development (structural constituents of ribosome (20-fold), H/ACA ribonucleoprotein complex subunit (18-fold), DNA binding and nucleosome assembly protein (5.3-fold), and prefoldin (5-fold)). These results indicate that exposure to P. putida can stimulate the growth of T. aggressivum and this interaction may be an important factor in increasing green mould disease in mushroom crops and so reducing yield.

Proteomic profiles during adventitious rooting of Eucalyptus species relevant to the cellulose industry

Adventitious rooting (AR) is an obligatory step for vegetative propagation of commercial woody species. Paper industries have interest in Eucalyptus globulus Labill and its hybrids due to low lignin and lipid contents, which facilitate cellulose extraction. However, this species and some of its hybrids are recalcitrant to rooting, often requiring exogenous auxin supply. Here we performed a comparative analysis of proteome changes during AR of E. globulus and the easy-to-root species Eucalyptus grandis W. Hill and the effects of exogenous auxin in different phases of the process (induction and formation), using a label-free quantification method. We identified 398 differentially abundant proteins, which were predicted to be involved in different biological pathways, mainly oxidative stress, energy metabolism and photosynthesis. Notable differences between species included proteins involved in oxidative stress, carbon and secondary metabolism. Exogenous auxin appeared to affect the availability of carbon sources and other phytohormones, besides cell cycle and microtubule—related proteins. Important players were also identified in each phase of AR. This is the first in depth analysis of protein changes during AR in Eucalyptus. The findings can be used in future studies to evaluate rooting competence in different genotypes and provide leads for AR improvement.

Comparative analyses of the variation of the transcriptome and proteome of Rhodobacter sphaeroides throughout growth

BackgroundIn natural environments, bacteria must frequently cope with extremely scarce nutrients. Most studies focus on bacterial growth in nutrient replete conditions, while less is known about the stationary phase. Here, we are interested in global gene expression throughout all growth phases, including the adjustment to deep stationary phase.ResultsWe monitored both the transcriptome and the proteome in cultures of the alphaproteobacterium Rhodobacter sphaeroides, beginning with the transition to stationary phase and at different points of the stationary phase and finally during exit from stationary phase (outgrowth) following dilution with fresh medium. Correlation between the transcriptomic and proteomic changes was very low throughout the growth phases. Surprisingly, even in deep stationary phase, the abundance of many proteins continued to adjust, while the transcriptome analysis revealed fewer adjustments. This pattern was reversed during the first 90 min of outgrowth, although this depended upon the duration of the stationary phase. We provide a detailed analysis of proteomic changes based on the clustering of orthologous groups (COGs), and compare these with the transcriptome.ConclusionsThe low correlation between transcriptome and proteome supports the view that post-transcriptional processes play a major role in the adaptation to growth conditions. Our data revealed that many proteins with functions in transcription, energy production and conversion and the metabolism and transport of amino acids, carbohydrates, lipids, and secondary metabolites continually increased in deep stationary phase. Based on these findings, we conclude that the bacterium responds to sudden changes in environmental conditions by a radical and rapid reprogramming of the transcriptome in the first 90 min, while the proteome changes were modest. In response to gradually deteriorating conditions, however, the transcriptome remains mostly at a steady state while the bacterium continues to adjust its proteome. Even long after the population has entered stationary phase, cells are still actively adjusting their proteomes.

Impact of Radiation on Tumor Derived Exosome Production, Proteomics and Biodistribution

Exosomes are cell derived nanoparticles implicated in malignant processes, including the determination of organ specific metastasis. This study aims to test the hypothesis that radiation interferes with the ability of tumor-derived exosomes to establish a pre-metastatic niche by affecting exosome production and proteomics. To understand the effect of radiation across tumors with different radiation sensitivities, breast cancer cell lines with known organotropism (MDA-MB-231 parental and the lung tropic subline, 4175) were compared to melanoma cancer cell line (B16F10) and exposed to 2 Gy vs. 10 Gy radiation. Exosomes were harvested from conditioned media using differential centrifugation and characterized using protein BCA, NanoSight, and electron microscopy. Exosome size was further characterized using a novel asymmetric flow fractionator. DNA packaging was quantified with Qubit ds DNA assay kit and proteomics analyzed through mass spectrometry. Biodistribution was assessed in vivo through retro-orbital sinus injection of fluorescently labeled exosomes and subsequent imaging of organ distribution. Radiation increased the quantity of exosomes produced in a dose dependent manner. Across all cell lines, 2 Gy vs. 10 Gy radiation increased exosome yield (ug/cell) by an average of 2.1 ± 0.4 and 7.8 ± 2.7-fold, respectively. In radioresistant melanoma cells, 2 Gy and 10 Gy radiation increased DNA content per exosome by 1.9 and 8.2-fold, respectively, whereas in more radiosensitive breast cancer cells, 2 Gy and 10 Gy only moderately increased DNA packaging by 1.1 and 1.4-fold. In general, radiation decreased exosome particle size. In breast cancer cells, mean particle size decreased from 135±6.9 nm to 125.7±1.8 and 124.3±3.9 nm for 2 Gy and 10 Gy radiation, respectively. Radiation also decreased the abundance of the smallest exosome subparticles, exomeres, which preferentially traffic to the liver to effect changes in metabolomics. For breast cancer cell lines, radiated exosomes retained the ability to traffic to the lung and liver 24 hours after injection. Pathway analysis of proteomic changes across radiation subgroups implicate significant changes in integrin signaling and remodeling of epithelial adherens junctions, including integrins and metalloproteases. Radiation significantly increased the quantity and DNA packaging of exosomes across all tumor cell lines. Although radiated exosomes trafficked to potential metastatic sites 24 hours after injection, their function in establishing a pre-metastatic niche remains to be explored. Changes in proteomics and exosome subpopulations suggest differences in migration and liver education. These changes trended with radiation dose and intrinsic radiation sensitivity, offering a promising platform for monitoring treatment response and underlying tumor biology in patients.

Quantitative Proteomic Analysis of Changes Related to Age and Calorie Restriction in Rat Liver Tissue.

Calorie restriction (CR) is the most frequently studied mechanism for increasing longevity, protecting against stress, and delaying age-associated diseases. Most studies have initiated CR in young animals to determine the protective effects against aging. Although aging phenomena are well-documented, the molecular mechanisms of aging and CR remain unclear. In this study, we observe changes in hepatic proteins upon age-related and diet-restricted changes in the rat liver using quantitative proteomics. Quantitative proteomes are measured using tandem mass tag labeling followed by LC-MS/MS. We compare protein levels in livers from young (6 months old) and old (25 months old) rats with 40% calorie-restricted (YCR and OCR, respectively) or ad libitum diets. In total, 44279 peptides and 3134 proteins are identified and 260 differentially expressed proteins are found. Functional enrichment analysis show that these proteins are mainly involved in glucose and fatty acid metabolism-related processes, consistent with the theory that energy metabolism regulation is dependent on age-related and calorie-restricted changes in liver tissue. In addition, proteins mediating inflammation and gluconeogenesis are increased in OCR livers, but not YCR livers. These results show that CR in old rats might not have antiaging benefits because liver inflammation is increased.

Analysis of proteomic changes in MDA-MB-231 cells induced by selected triorganotin compounds, biologically active ligands of nuclear retinoid X receptors

Analysis of proteomic changes in MDA-MB-231 cells induced by selected triorganotin compounds, biologically active ligands of nuclear retinoid X receptors

Accessory subunits are integral for assembly and function of human mitochondrial complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.