Proteomic Landscape Research Articles

Unraveling the Proteomic Landscape of Intestinal Epithelial Cell-Derived Exosomes in Mice.

PurposeThis study aimed to identify the biological functions of small intestine intestinal epithelial cell derived exosomes (IEC-Exos) and further distinguished the difference proteins in IEC-Exos between ileum and jejunum related to function of the digestive system and occurrence of several diseases.Materials and MethodsIECs of Male C57BL/6J mice were isolated. IEC-Exos were extracted from jejunum and ileum epithelial cell culture fluid by ultracentrifugation. In addition, isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to detect IEC-Exo proteins and conduct biological information analysis.ResultsThe results showed that compared with jejunum IEC-Exos from ileum IEC-Exos, there were 393 up-regulated proteins and 346 down-regulated proteins. IECs-Exos, especially derived from jejunum, were rich in angiotensin-converting enzyme 2 (ACE2). The highly expressed proteins from ileum IEC-Exos were mostly enriched in genetic information processing pathways, which mainly mediate the processes of bile acid transport, protein synthesis and processing modification. In contrast, the highly expressed proteins from jejunum IEC-Exos were mainly enriched in metabolic pathways involved in sugar, fatty acid, amino acid, drug, and bone metabolism, etc. The differentially expressed proteins between ileum and jejunum IEC-Exos were not only related to the function of the digestive system but also closely related to the occurrence of infectious diseases, endocrine diseases and osteoarthritis, etc.ConclusionIEC-Exos there were many differentially expressed proteins between ileum and jejunum, which played different roles in regulating intestinal biological functions. ACE2, the main host cell receptor of SARS-CoV-2, was highly expressed in IEC-Exos, which indicated that IEC-Exos may be a potential route of SARS-CoV-2 infection.

The power of chromosome-scale, haplotype-resolved genomes

The assembly of chromosome-scale and haplotype-resolved reference genomes is now more easily attainable, largely due to various improvements in both assembly algorithms and long-read sequencing technologies (see recent review by Michael and VanBuren, 2020 Michael T.P. VanBuren R. Building near-complete plant genomes. Curr. Opin. Plant Biol. 2020; 54: 26-33 Google Scholar ). Due to these technological advancements, there has been a shift away from using highly inbred accessions, in some instances double haploids, to instead assembling the genomes of important reference genotypes. For example, a haplotype-resolved genome was recently assembled for an important cultivated garden strawberry (Fragaria x ananassa) cultivar Royal Royce, which is not only highly heterozygous but also octoploid (8×) ( Hardigan et al., 2021 Hardigan M.A. Feldmann M.J. Pincot D.D.A. Famula R.A. Vachev M.V. Madera M.A. Zerbe P. Mars K. Peluso P. Rank D. et al. Blueprint for phasing and assembling the genomes of heterozygous polyploids: application to the octoploid genome of strawberry. Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.11.03.467115 Google Scholar ). Similarly, haplotype-phased genomes have been assembled for other important crops, including cassava (Manihot esculenta) ( Hu et al., 2021 Hu W. Ji C. Shi H. Liang Z. Ding Z. Ye J. Ou W. Zhou G. Tie W. Yan Y. et al. Allele-defined genome reveals biallelic differentiation during cassava evolution. Mol. Plant. 2021; 14: 851-854 Google Scholar ) and potato (Solanum tuberosum) ( Hoopes et al., 2022 Hoopes G. Meng X. Hamilton J.P. Achakkagari S.R. de Alves Freitas Guesdes F. Bolger M.E. Coombs J.J. Esselink D. Kaiser N.R. Kodde L. et al. Phased, chromosome-scale genome assemblies of tetraploid potato reveals a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity. Mol. Plant. 2022; (S1674-2052, 00003-X)https://doi.org/10.1016/j.molp.2022.01.003 Google Scholar ). This has resulted in the development of new genomic resources that can be directly used in guiding molecular breeding efforts, without worrying about the gene presence-absence variation that exists within a crop or species ( Golicz et al., 2016 Golicz A.A. Batley J. Edwards D. Towards plant pangenomics. Plant Biotechnol. J. 2016; 14: 1099-1105 Google Scholar ; Bayer et al., 2021 Bayer P.E. Scheben A. Golicz A.A. Yuan Y. Faure S. Lee H. Chawla H.S. Anderson R. Bancroft I. Raman H. et al. Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids. Plant Biotechnol. J. 2021; 19: 2488-2500 Google Scholar ). An additional benefit of having a reference genome of an important cultivar and/or genotype, versus from a highly inbred line, is that valuable insights that are potentially gained from the analysis of genetic variants present among haplotypes are not lost, allowing for additional insights.

Integrative proteomic and phosphoproteomic profiling of invasive micropapillary breast carcinoma

Invasive micropapillary carcinoma (IMPC) is a rare subtype of breast cancer with an aggressive phenotype and a poor prognosis. The mechanism of tumorigenesis of IMPC in breast remains unknown. Integrative analysis of the proteome and phosphoproteome may shed light on the mechanism of IMPC carcinogenesis. In our study, primary IMPC and paired normal breast tissue were collected from six patients and subjected to label free LC-MS/MS for quantitative proteomic and phosphoproteomic analysis. Kinase-substrate enrichment analysis (KSEA) was conducted to identify hyperactivated/inhibited kinases. Proteomic and phosphoproteomic data was combined to investigate cancer specific activated pathways. A total of 1331 differentially expressed proteins were identified. The proteomic analysis revealed a dysregulation of protein homeostasis in IMPC. Phosphoproteomic profiling identified 856 differentially phosphorylated phosphosites in 655 proteins. KSEA found that cyclin dependent kinases (CDKs) and the p90 ribosomal S6 kinases (RSKs) were highly activated, while protein kinase A (PKA) and protein kinase C (PKC) families were significantly inhibited in IMPC. Finally, cancer-specific activation of mTORC1/S6K2 signaling was also discerned in our integrative analysis. Overall, this study provides a comprehensive landscape of the proteome and phosphoproteome of IMPC, which will contribute to a further understanding of IMPC tumorigenesis and treatment. SignificanceThis is the first study to provide the integrative proteomic and phosphoproteomic landscape of IMPC. Our study demonstrated that protein homeostasis dysregulation is one of the most prominent characteristics in IMPC. We also identifying several kinases which might have the potential to be novel targets in clinical practice. Cancer-specific activation of mTORC1/S6K2 signaling was discerned by integrative proteomic and phosphoproteomic analysis. The present study might contribute to a further understanding of IMPC tumorigenesis and treatment.

The proteomic landscape of ovarian cancer cells in response to melatonin

Ovarian cancer (OC) is the most lethal gynecological malignancy with a highly negative prognosis. Melatonin is an indoleamine secreted by the pineal gland during darkness and has shown antitumor activity in both in vitro and in vivo experiments. Herein, we investigated the influence of melatonin on the proteome of human ovarian carcinoma cells (SKOV-3 cell line) using the Ultimate 3000 LC Liquid NanoChromatography equipment coupled to a Q-Exactive mass spectrometry. After 48 h of treatment, melatonin induced a significant cytotoxicity especially with the highest melatonin concentration. The proteomic profile revealed 639 proteins in the control group, and 98, 110, and 128 proteins were altered by melatonin at the doses of 0.8, 1.6, and 2.4 mM, respectively. Proteins associated with the immune system and tricarboxylic acid cycle were increased in the three melatonin-exposed groups of cells. Specifically, the dose of 2.4 mM led to a reduction in molecules associated with protein synthesis, especially those of the ribosomal protein family. We also identified 28 potential genes shared between normal ovarian tissue and OC in all experimental groups, and melatonin was predicted to alter genes encoding ribosomal proteins. Notably, the set of proteins changed by melatonin was linked to a better prognosis for OC patients. We conclude that melatonin significantly alters the proteome of SKOV-3 cells by changing proteins involved with the immune response and mitochondrial metabolism. The concentration of 2.4 mM of melatonin promoted the largest number of protein changes. The evidence suggests that melatonin may be an effective therapeutic strategy against OC.

Clinical relevance of proteomic profiling in de novo pediatric acute myeloid leukemia: a Children's Oncology Group study.

Pediatric acute myeloid leukemia (AML) remains a fatal disease for at least 30% of patients, stressing the need for improved therapies and better risk stratification. As proteins are the unifying feature of (epi)genetic and environmental alterations, and are often targeted by novel chemotherapeutic agents, we studied the proteomic landscape of pediatric AML. Protein expression and activation levels were measured in 500 bulk leukemic patients’ samples and 30 control CD34+ cell samples, using reverse phase protein arrays with 296 strictly validated antibodies. The multistep MetaGalaxy analysis methodology was applied and identified nine protein expression signatures (PrSIG), based on strong recurrent protein expression patterns. PrSIG were associated with cytogenetics and mutational state, and with favorable or unfavorable prognosis. Analysis based on treatment (i.e., ADE vs. ADE plus bortezomib) identified three PrSIG that did better with ADE plus bortezomib than with ADE alone. When PrSIG were studied in the context of cytogenetic risk groups, PrSIG were independently prognostic after multivariate analysis, suggesting a potential value for proteomics in combination with current classification systems. Proteins with universally increased (n=7) or decreased (n=17) expression were observed across PrSIG. Certain proteins significantly differentially expressed from normal could be identified, forming a hypothetical platform for personalized medicine.

Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder’s efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.

Intercellular Cardiomyocyte to Cardiac Fibroblast Signalling Regulates Global Proteome and Phosphoproteome Landscape to Promote Oxidative Stress via CK2

A hallmark of pathological cardiac remodelling is cardiac fibrosis, a process regulated by Angiotensin II (Ang II) and/or TGFβ-driven crosstalk between cardiomyocytes and cardiac fibroblasts. As direct therapeutic targeting of Ang II/TGFβ for cardiac fibrosis remains challenging, discovery of common downstream effectors presents an opportunity for novel therapeutic strategy.

Proteomic Landscape of Primary and Metastatic Brain Tumors for Heterogeneity Discovery

Proteomic Landscape of Primary and Metastatic Brain Tumors for Heterogeneity Discovery

System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy.

Rationale: Diabetic retinopathy (DR) is a major complication of diabetes mellitus causing significant vision loss. DR is a multifactorial disease involving changes in retinal microvasculature and neuronal layers, and aberrations in vascular endothelial growth factors (VEGF) and inflammatory pathways. Despite the success of anti-VEGF therapy, many DR patients do not respond well to the treatment, emphasizing the involvement of other molecular players in neuronal and vascular aberrations in DR.Methods: We employed advanced mass spectrometry-based proteome profiling to obtain a global snapshot of altered protein abundances in vitreous humor from patients with proliferative DR (PDR) in comparison to individuals with epiretinal membrane without active DR or other retinal vascular complications. Global proteome correlation map and protein-protein interaction networks were used to probe into the functional inclination of proteins and aberrated molecular networks in PDR vitreous. In addition, peptide-centric analysis of the proteome data was carried out to identify proteolytic processing, primarily ectodomain shedding events in PDR vitreous. Functional validation experiments were performed using preclinical models of ocular angiogenesis.Results: The vitreous proteome landscape revealed distinct dysregulations in several metabolic, signaling, and immune networks in PDR. Systematic analysis of altered proteins uncovered specific impairment in ectodomain shedding of several transmembrane proteins playing critical roles in neurodegeneration and angiogenesis, pointing to defects in their regulating sheddases, particularly ADAM10, which emerged as the predominant sheddase. We confirmed that ADAM10 protease activity was reduced in animal models of ocular angiogenesis and established that activation of ADAM10 can suppress endothelial cell activation and angiogenesis. Furthermore, we identified the impaired ADAM10-AXL axis as a driver of retinal angiogenesis.Conclusion: We demonstrate restoration of aberrant ectodomain shedding as an effective strategy for treating PDR and propose ADAM10 as an attractive therapeutic target. In all, our study uncovered impaired ectodomain shedding as a prominent feature of PDR, opening new possibilities for advancement in the DR therapeutic space.

Proteomic Landscape of Primary and Metastatic Brain Tumors for Heterogeneity Discovery

Proteomic Landscape of Primary and Metastatic Brain Tumors for Heterogeneity Discovery

Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk.

Background:Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied.Objective:This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis.Methods:Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point.Results:At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions.Discussion:These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676

Quantitative proteomics reveals differential immunoglobulin-associated proteome (IgAP) in patients of acute myocardial infarction and chronic coronary syndromes

B cells and immunoglobulins are implicated in the pathogenesis of chronic diseases, including coronary artery disease (CAD). However, it remains elusive how the humoral immunity is incriminated in the disease progression of CAD. Using serum samples of chronic coronary syndrome (CCS) and acute myocardial infarction (AMI), we conducted a quantitative profiling of the proteomic landscape recognized by immunoglobulins, which we term immunoglobulin-associated proteome (IgAP). Intriguingly, CCS and AMI patients displayed distinctive IgAP profiles that enriched proteins in the pathways of blood coagulation regulation and lipoprotein transport, suggesting that CCS-AMI transition involves changes of these pathways that are associated with immunoglobulins. Furthermore, we identified immunoglobulin-bound coagulation factor X (F10) as a potential biomarker and validated it with an independent cohort of CCS, AMI and healthy individuals. Our study indicates that IgAP proteins may serve as novel diagnostic biomarkers for CCS and AMI. SignificanceOur work it demonstrates a clear implication of immunoglobulin-associated proteome (IgAP), the proteomic landscape recognized by immunoglobulins, in the pathogenesis of CAD. In addition, it reports for the first time that immunoglobulin-bound F10 is implicated in CAD.

Proteomic dissection of rice cytoskeleton reveals the dominance of microtubule and microfilament proteins, and novel components in the cytoskeleton-bound polysome

The plant cytoskeleton persistently undergoes remodeling to achieve its roles in supporting cell division, differentiation, cell expansion and organelle transport. However, the links between cell metabolism and cytoskeletal networks, particularly how the proteinaceous components execute such processes remain poorly understood. We investigated the cytoskeletal proteome landscape of rice to gain better understanding of such events. Proteins were extracted from highly enriched cytoskeletal fraction of four-week-old rice seedlings, and the purity of the fraction was stringently monitored. A total of 2577 non-redundant proteins were identified using both gel-based and gel-free approaches, which constitutes the most comprehensive dataset, thus far, for plant cytoskeleton. The data set includes both microtubule and microfilament-associated proteins and their binding proteins comprising hypothetical as well as novel cytoskeletal proteins. Further, various in-silico analyses were performed, and the proteins were functionally classified on the basis of their gene ontology. The catalogued proteins were validated through their sequence analysis. Extensive comparative analysis of our dataset with the non-redundant set of cytoskeletal proteins across plant species affirms unique as well as overlapping candidates. Together, these findings unveil new insights of how cytoskeletons undergo dynamic remodeling in rice to drive seedling development processes in rapidly changing in planta environment.

Proteomic profiling identifies signatures associated with progression of precancerous gastric lesions and risk of early gastric cancer.

BackgroundMolecular features underlining the multistage progression of gastric lesions and development of early gastric cancer (GC) are poorly understood, restricting the ability to GC prevention and management.MethodsWe portrayed proteomic landscape and explored proteomic signatures associated with progression of gastric lesions and risk of early GC. Tissue proteomic profiling was conducted for a total of 324 subjects. A case-control study was performed in the discovery stage (n=169) based on populations from Linqu, a known high-risk area for GC in China. We then conducted two-stage validation, including a cohort study from Linqu (n = 56), with prospective follow-up for progression of gastric lesions (280–473 days), and an independent case-control study from Beijing (n = 99).FindingsThere was a clear distinction in proteomic features for precancerous gastric lesions and GC. We derived four molecular subtypes of gastric lesions and identified subtype-S4 with the highest progression risk. We found 104 positively-associated and 113 inversely-associated proteins for early GC, with APOA1BP, PGC, HPX and DDT associated with the risk of gastric lesion progression. Integrating these proteomic signatures, the ability to predict progression of gastric lesions was significantly strengthened (areas-under-the-curve=0.88 (95%CI: 0.78–0.99) vs. 0.56 (0.36–0.76), Delong's P = 0.002). Immunohistochemistry assays and examination at mRNA level validated the findings for four proteins.InterpretationWe defined proteomic signatures for progression of gastric lesions and risk of early GC, which may have translational significance for identifying particularly high-risk population and detecting GC at an early stage, improving potential for targeted GC prevention.FundingThe funders are listed in the Acknowledgement.

CSIG-27. PROTEOMIC ANALYSIS OF GENETICALLY STRATIFIED MENINGIOMA

Abstract WHO grade II and III as well as some WHO grade I meningioma are clinically aggressive. Approximately 60% sporadic meningiomas harbour mutations in the NF2 gene, others in genes including TRAF7, KLF4, AKT1, SMO and PIK3CA . However, the molecular mechanisms behind meningioma tumourigenesis are still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterising the proteomic landscape. We performed (phosho)proteomic profiling of grade I, II and III meningiomas and three different mutational groups: AKT1 E17K /TRAF7, KLF4 K409Q /TRAF7 and NF2 -/-. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry. Looking at all grades bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 and upregulation and activation of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins described included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, Hexokinase 2. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups with proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1 E17K /TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. Less proteins were commonly significantly upregulated in KLF4 K409Q /TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 were verified. Analyses of 6600 phospho-sites predicted regulatory kinases. Further validation and functional verification of potential candidates is ongoing.

Single-Cell Proteomics Identifies Leukemia Landscape Associated with Clinical Outcomes in R/R AML Treated with MDM2i (Milademetan) and FLT3i (Quizartinib): Putative Role of CD68 and Diversity Index

Single-Cell Proteomics Identifies Leukemia Landscape Associated with Clinical Outcomes in R/R AML Treated with MDM2i (Milademetan) and FLT3i (Quizartinib): Putative Role of CD68 and Diversity Index

Identification of a Highly Deregulated eIF4F Translation Initiation Complex in Drug-Resistant BCR-ABL + Cells By a Phospho-Proteomic Antibody Microarray

Protein-tyrosine kinase inhibitors (TKIs) have been effective for treatment of early stages of chronic myeloid leukemia (CML). However, BCR-ABL-dependent resistance mechanisms and TKI-unresponsive quiescent leukemic stem cells (LSCs) can result in drug resistance and disease relapse. We have demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs. It interacts with BCR-ABL through the AHI-1 WD40 domain and with other proteins, like dynamin-2 (DNM2), through its SH3 domain, to enhance leukemic-initiating activities and TKI resistance. To uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance in CML stem/progenitor cells, an advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL + cells co-transduced with wild-type Ahi-1 or Ahi-1 SH3 Δ mutant in the presence or absence of imatinib (IM). The microarray simultaneously quantified the differences in expression and phosphorylation sites of proteins in multiple signaling pathways using 1325 antibodies in duplicate measurements, and each microarray analysis was performed in duplicate. Significant changes in antibody signals for protein expression or phosphorylation were determined using limma. This analysis revealed that the overexpression of wild-type Ahi-1 (WT Ahi-1) has a profound differential effect, compared to the SH3 domain deleted Ahi-1 (Ahi-1 SH3 Δ), on the expression and phosphorylation status of proteins in BCR-ABL + cells with and without IM treatment. BCR-ABL + cells co-expressing WT Ahi-1 had a greater number of significantly differential antibody signals (7 increases, 42 decreases) when compared to BCR-ABL + cells, while Ahi-1 SH3 Δ expressing cells resulted in fewer significantly differential antibody signals (12 increases, 2 decreases) compared to BCR-ABL + cells. IM treatment resulted in WT Ahi-1 expressing cells having the greatest number of significantly differential antibody signals (7 increases, 56 decreases) compared to BCR-ABL + cells (5 decreases) and those co-expressing Ahi-1 SH3 Δ (2 increases, 9 decreases). Pathway enrichment analysis, using gProfiler, identified that the targets with significantly increased differential antibody signal after IM treatment in WT Ahi-1 cells were related to the regulation of translation initiation complex (p>0.0001). Interestingly, our RNA-seq dataset analysis further identified several members of the eukaryotic initiation factor 4F (eIF4F) complex to be significantly upregulated in CD34 + CML patient cells compared to normal bone marrow, particularly eIF4G1, the scaffold protein of the eIF4F complex involved in translation initiation (2-fold, p=0.001), as well as mTOR, a key regulator that controls the assembly of the eIF4F complex. This finding prompted us to further explore the regulation of translation initiation and the members of the eIF4F complex in BCR-ABL + cells. Immunoblotting demonstrated that BCR-ABL + cells co-transduced with WT Ahi-1 showed increased expression of eIF4G1 (3-fold) and eIF4B (2-fold), a cofactor that regulates the helicase activity of the eIF4F complex, compared to BCR-ABL + cells. Additionally, Cyclin D3, a gene reported to be sensitive to eIF4F translational activity, was found to have slightly increased expression (1.4-fold) in WT Ahi-1 cells compared to BCR-ABL + cells. Most interestingly, these results were also demonstrated in IM-resistant CML cells as compared to IM-sensitive cells, with an increase in eIF4G1 expression (2-fold), phosphorylation of eIF4B (5-fold, p=0.003), and Cyclin D3 expression (4-fold, p<0.05). Mechanistically, eIF4G1 knockdown by shRNA impaired survival (5-fold, p<0.0001) and increases TKI sensitivity in IM-resistant cells (3-fold, p<0.05). A new protein interaction between eIF4G1 and the mRNA cap-binding protein eIF4E was further identified in these cells using a proximity ligation assay. Thus, we have uncovered that the eIF4F complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, has increased activity in IM-resistant cells, which may contribute to the regulation of TKI resistance in CML. DisclosuresNo relevant conflicts of interest to declare.

14-3-3ζ negatively regulates mitochondrial biogenesis in GBM residual cells

Glioblastoma (GBM) is the most lethal primary brain tumour with a median survival of only 15 months. We have previously demonstrated the generation of an in vitro therapy resistance model that captures the residual resistant (RR) disease cells of GBM post-radiation. We also reported the proteomic landscape of parent, residual, and relapse cells using iTRAQ based quantitative proteomics of glioma cells. The proteomics data revealed significant up-regulation (fold change >1.5) of 14-3-3ζ, specifically in GBM RR cells. This was further confirmed by western blots in residual cells generated from GBM cell lines and patient sample-derived short-term primary culture. ShRNA-mediated knockdown of 14-3-3ζ radio-sensitized GBM cells and further stimulated therapy-induced senescence (TIS) and multinucleated giant cells (MNGCs) phenotype in RR cells. Intriguingly, 14-3-3ζ knockdown residual cells also showed a significantly higher number of mitochondria and increased mtDNA content. Indeed, in vitro GST pull-down mass spectrometry analysis of GST tagged 14-3-3ζ from RR cells identified novel interacting partners of 14-3-3ζ involved in cellular metabolism. Taken together, here we identified novel interacting partners of 14-3-3ζ and proposed an unconventional function of 14-3-3ζ as a negative regulator of TIS and mitochondrial biogenesis in residual resistant cells and loss of which also radio-sensitize GBM cells.

The promise of organoids for unraveling the proteomic landscape of the developing human brain.

Cerebral organoids offer an opportunity to bioengineer experimental avatars of the developing human brain and have already begun garnering relevant insights into complex neurobiological processes and disease. Thus far, investigations into their heterogeneous cellular composition and developmental trajectories have been largely limited to transcriptional readouts. Recent advances in global proteomic technologies have enabled a new range of techniques to explore dynamic and non-overlapping spatiotemporal protein-level programs operational in these humanoid neural structures. Here we discuss these early protein-based studies and their potentially essential role for unraveling critical secreted paracrine signals, processes with poor proteogenomic correlations, or neurodevelopmental proteins requiring post-translational modification for biological activity. Integrating emerging proteomic tools with these faithful human-derived neurodevelopmental models could transform our understanding of complex neural cell phenotypes and neurobiological processes, not exclusively driven by transcriptional regulation. These insights, less accessible by exclusive RNA-based approaches, could reveal new knowledge into human brain development and guide improvements in neural regenerative medicine efforts.

Inhibition of hypoxia-induced Mucin 1 alters the proteomic composition of human osteoblast-produced extracellular matrix, leading to reduced osteogenic and angiogenic potential.

The bone microenvironment is one of the most hypoxic regions of the human body and in experimental models; hypoxia inhibits osteogenic differentiation of mesenchymal stromal cells (MSCs). Our previous work revealed that Mucin 1 (MUC1) was dynamically expressed during osteogenic differentiation of human MSCs and upregulated by hypoxia. Upon stimulation, its C‐terminus (MUC1‐CT) is proteolytically cleaved, translocases to the nucleus, and binds to promoters of target genes. Therefore, we assessed the MUC1‐mediated effect of hypoxia on the proteomic composition of human osteoblast‐derived extracellular matrices (ECMs) and characterized their osteogenic and angiogenic potentials in the produced ECMs. We generated ECMs from osteogenically differentiated human MSC cultured in vitro under 20% or 2% oxygen with or without GO‐201, a MUC1‐CT inhibitor. Hypoxia upregulated MUC1, vascular endothelial growth factor, and connective tissue growth factor independent of MUC1 inhibition, whereas GO‐201 stabilized hypoxia‐inducible factor 1‐alpha. Hypoxia and/or MUC1‐CT inhibition reduced osteogenic differentiation of human MSC by AMP‐activated protein kinase/mTORC1/S6K pathway and dampened their matrix mineralization. Hypoxia modulated ECMs by transforming growth factor‐beta/Smad and phosphorylation of NFκB and upregulated COL1A1, COL5A1, and COL5A3. The ECMs of hypoxic osteoblasts reduced MSC proliferation and accelerated their osteogenic differentiation, whereas MUC1‐CT‐inhibited ECMs counteracted these effects.In addition, ECMs generated under MUC1‐CT inhibition reduced the angiogenic potential independent of oxygen concentration. We claim here that MUC1 is critical for hypoxia‐mediated changes during osteoblastogenesis, which not only alters the proteomic landscape of the ECM but thereby also modulates its osteogenic and angiogenic potentials.