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1.
Lab Invest ; 103(4): 100052, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36870295

RESUMO

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Antígeno B7-H1 , Cadeias alfa de HLA-DR , Inclusão em Parafina/métodos , Hematoxilina , Amarelo de Eosina-(YS) , Proteínas/metabolismo , Peptídeos , Biomarcadores , Espectrometria de Massas em Tandem/métodos , Formaldeído/química , Fixação de Tecidos
2.
J Cell Sci ; 134(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34406412

RESUMO

In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.


Assuntos
Receptores ErbB , Transdução de Sinais , Epirregulina/genética , Epirregulina/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Fosforilação
3.
Lab Invest ; 102(10): 1101-1108, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35595825

RESUMO

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.


Assuntos
Neoplasias da Mama , Imunoconjugados , Neoplasias da Mama/genética , Feminino , Humanos , Receptor ErbB-2/análise , Receptor ErbB-2/genética
4.
Mol Cell ; 40(6): 893-904, 2010 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-21172655

RESUMO

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3⁻/⁻ livers at 3 months of age. Livers of Sirt3⁻/⁻ mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3⁻/⁻ MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD⁻/⁻ MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3⁻/⁻ MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3⁻/⁻ livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.


Assuntos
Sequência Conservada , Evolução Molecular , Lisina/metabolismo , Estresse Oxidativo , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismo , Acetilação , Animais , Arginina/metabolismo , Linhagem Celular , Camundongos , Mutagênese Sítio-Dirigida , Sirtuína 3/deficiência , Sirtuína 3/genética
5.
J Proteome Res ; 16(3): 1364-1375, 2017 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-28088864

RESUMO

An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10 000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.


Assuntos
Células/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Redes e Vias Metabólicas , Apoptose , Linhagem Celular , Sobrevivência Celular , Cisplatino/farmacologia , Biologia Computacional/métodos , Humanos
6.
Hepatol Res ; 47(13): 1469-1483, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28258704

RESUMO

BACKGROUND: Molecular signaling events associated with the necroinflammatory changes in nonalcoholic steatohepatitis (NASH) are not well understood. AIMS: To understand the molecular basis of NASH, we evaluated reversible phosphorylation events in hepatic tissue derived from Class III obese subjects by phosphoproteomic means with the aim of highlighting key regulatory pathways that distinguish NASH from non-alcoholic fatty liver disease (also known as simple steatosis; SS). MATERIALS & METHODS: Class III obese subjects undergoing bariatric surgery underwent liver biopsy (eight normal patients, eight with simple steatosis, and eight NASH patients). Our strategy was unbiased, comparing global differences in liver protein reversible phosphorylation events across the 24 subjects. RESULTS: Of the 3078 phosphorylation sites assigned (2465 phosphoserine, 445 phosphothreonine, 165 phosphotyrosine), 53 were altered by a factor of 2 among cohorts, and of those, 12 were significantly increased or decreased by ANOVA (P < 0.05). DISCUSSION: Statistical analyses of canonical signaling pathways identified carbohydrate metabolism and RNA post-transcriptional modification among the most over-represented networks. CONCLUSION: Collectively, these results raise the possibility of abnormalities in carbohydrate metabolism as an important trigger for the development of NASH, in parallel with already established abnormalities in lipid metabolism.

7.
Gastroenterology ; 146(7): 1739-51.e14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24530706

RESUMO

BACKGROUND & AIMS: The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS: SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS: SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS: In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.


Assuntos
Dano ao DNA , Células Epiteliais/enzimologia , Receptores ErbB/metabolismo , Mucosa Gástrica/enzimologia , Infecções por Helicobacter/enzimologia , Helicobacter pylori/metabolismo , Receptor ErbB-2/metabolismo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Colômbia , Progressão da Doença , Ativação Enzimática , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Receptores ErbB/deficiência , Receptores ErbB/genética , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/enzimologia , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Honduras , Humanos , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fosforilação , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Análise de Componente Principal , Multimerização Proteica , Receptor ErbB-2/genética , Transdução de Sinais , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Tennessee , Poliamina Oxidase
8.
J Proteome Res ; 13(7): 3303-13, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24874604

RESUMO

Asef2, a 652-amino acid protein, is a guanine nucleotide exchange factor (GEF) that regulates cell migration and other processes via activation of Rho family GTPases, including Rac. Binding of the tumor suppressor adenomatous polyposis coli (APC) to Asef2 is known to induce its GEF activity; however, little is currently known about other modes of Asef2 regulation. Here, we investigated the role of phosphorylation in regulating Asef2 activity and function. Using high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS), we obtained complete coverage of all phosphorylatable residues and identified six phosphorylation sites. One of these, serine 106 (S106), was particularly intriguing as a potential regulator of Asef2 activity because of its location within the APC-binding domain. Interestingly, mutation of this serine to alanine (S106A), a non-phosphorylatable analogue, greatly diminished the ability of Asef2 to activate Rac, while a phosphomimetic mutation (serine to aspartic acid, S106D) enhanced Rac activation. Furthermore, expression of these mutants in HT1080 cells demonstrated that phosphorylation of S106 is critical for Asef2-promoted migration and for cell-matrix adhesion assembly and disassembly (adhesion turnover), which is a process that facilitates efficient migration. Collectively, our results show that phosphorylation of S106 modulates Asef2 GEF activity and Asef2-mediated cell migration and adhesion turnover.


Assuntos
Adesão Celular , Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação , Serina/química
9.
J Am Chem Soc ; 136(34): 11864-6, 2014 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-25099620

RESUMO

Lipid electrophiles modify cellular targets, altering their function. Here, we describe histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells: four 4-ketoamides to Lys and three Michael adducts to His. A 4-ketoamide adduct residing at H3K27 was identified in stimulated macrophages. Modification of histones H3 and H4 prevented nucleosome assembly.


Assuntos
Aldeídos/química , Adutos de DNA/química , Epigênese Genética/fisiologia , Histonas/química , Estresse Oxidativo/fisiologia , Processamento de Proteína Pós-Traducional , Acilação , Aldeídos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Adutos de DNA/genética , Adutos de DNA/metabolismo , Epigênese Genética/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Estresse Oxidativo/genética
10.
PLoS Pathog ; 7(9): e1002237, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21909278

RESUMO

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5ß1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.


Assuntos
Proteínas de Bactérias/fisiologia , Fímbrias Bacterianas/fisiologia , Ilhas Genômicas , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Estômago/microbiologia
11.
Proteomics ; 12(2): 329-38, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22106071

RESUMO

Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution-phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. Nearly, 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources.


Assuntos
Exossomos/química , Proteoma/análise , Proteômica/métodos , Urina/química , Adulto , Biomarcadores/análise , Biomarcadores/química , Biomarcadores/urina , Biologia Computacional , Bases de Dados de Proteínas , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Exossomos/ultraestrutura , Feminino , Humanos , Masculino , Microscopia Eletrônica , Proteoma/química , Espectrometria de Massas em Tandem , Adulto Jovem
12.
J Lipid Res ; 53(3): 379-389, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22215797

RESUMO

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Obesidade/sangue , Obesidade/metabolismo , Animais , Apolipoproteína C-II/sangue , Apolipoproteínas A/sangue , Western Blotting , Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol/genética , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Dieta Hiperlipídica/efeitos adversos , Feminino , Hiperinsulinismo/sangue , Hiperinsulinismo/induzido quimicamente , Insulina/sangue , Lipoproteínas HDL/sangue , Lipoproteínas VLDL/sangue , Camundongos , Camundongos Transgênicos , Obesidade/genética , Ovariectomia , Triglicerídeos/sangue , Aumento de Peso/efeitos dos fármacos
13.
J Clin Pathol ; 75(9): 636-642, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34353876

RESUMO

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.


Assuntos
Colite Ulcerativa , Biópsia , Colite Ulcerativa/patologia , Colonoscopia , Humanos , Interleucina-23 , Mucosa Intestinal/patologia , Proteômica , Índice de Gravidade de Doença
14.
Cell Rep ; 34(3): 108636, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33472061

RESUMO

The chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Descoberta de Drogas , Fase G2/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Terapia de Alvo Molecular , Ligação Proteica
15.
Nat Cell Biol ; 23(12): 1240-1254, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34887515

RESUMO

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Nanopartículas/metabolismo , Doença de Alzheimer/patologia , Enzima de Conversão de Angiotensina 2/metabolismo , Transporte Biológico/fisiologia , Biomarcadores/metabolismo , COVID-19/patologia , Doenças Cardiovasculares/patologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Ácido Láctico/metabolismo , MicroRNAs/genética , Nanopartículas/classificação , Neoplasias/patologia , Microambiente Tumoral
16.
Mol Cell Proteomics ; 7(9): 1651-67, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18504258

RESUMO

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.


Assuntos
Proteínas de Transporte/metabolismo , Exocitose , Proteínas/análise , Proteômica/métodos , Vesículas Transportadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia Líquida/métodos , Cães , Fluorescência , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Humanos , Espectrometria de Massas , Transporte Proteico/efeitos da radiação , Proteínas/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Vesículas Transportadoras/efeitos da radiação , Ácidos Tri-Iodobenzoicos/metabolismo
17.
Oncogene ; 39(22): 4465-4474, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32350444

RESUMO

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and ß-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and ß-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of ß-catenin signaling.


Assuntos
Adenocarcinoma/etiologia , Dano ao DNA , Gastrite/enzimologia , Infecções por Helicobacter/enzimologia , Helicobacter pylori/patogenicidade , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Espermina/metabolismo , Neoplasias Gástricas/etiologia , Adenocarcinoma/microbiologia , Animais , Transformação Celular Neoplásica , Gastrite/genética , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Proteoma , RNA Mensageiro/biossíntese , Transdução de Sinais , Espermidina/biossíntese , Neoplasias Gástricas/microbiologia , beta Catenina/fisiologia , Poliamina Oxidase
18.
Chem Res Toxicol ; 22(6): 1069-76, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19364102

RESUMO

1,2-Dibromoethane and 1,3-butadiene are cancer suspects present in the environment and have been used widely in industry. The mutagenic properties of 1,2-dibromoethane and the 1,3-butadiene oxidation product diepoxybutane are thought to be related to the bis-electrophilic character of these chemicals. The discovery that overexpression of O(6)-alkylguanine alkyltransferase (AGT) enhances bis-electrophile-induced mutagenesis prompted a search for other proteins that may act by a similar mechanism. A human liver screen for nuclear proteins that cross-link with DNA in the presence of 1,2-dibromoethane identified histones H2b and H3 as candidate proteins. Treatment of isolated histones H2b and H3 with diepoxybutane resulted in DNA-protein cross-links and produced protein adducts, and DNA-histone H2b cross-links were identified (immunochemically) in Escherichia coli cells expressing histone H2b. However, heterologous expression of histone H2b in E. coli failed to enhance bis-electrophile-induced mutagenesis. These results are similar to those found with the cross-link candidate glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [ Loecken , E. M. and Guengerich , F. P. ( 2008 ) Chem. Res. Toxicol. 21 , 453 - 458 ], but in contrast to GAPDH, histone H2b bound DNA with even higher affinity than AGT. The extent of DNA cross-linking of isolated histone H2b was similar to that of AGT, suggesting that differences in postcross-linking events explain the difference in mutagenesis.


Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Compostos de Epóxi/química , Histonas/química , Mutagênicos/química , Alquil e Aril Transferases/metabolismo , Reagentes de Ligações Cruzadas/toxicidade , DNA/metabolismo , Compostos de Epóxi/toxicidade , Histonas/metabolismo , Humanos , Mutagênese , Mutagênicos/toxicidade , Fragmentos de Peptídeos/análise , Proteínas Recombinantes/metabolismo
19.
mSphere ; 4(4)2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391278

RESUMO

Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. bruceiIMPORTANCETrypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.


Assuntos
Homeostase , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Adenilato Quinase/metabolismo , Mitocôndrias , Estresse Oxidativo , Fosforilação , Proteômica , Trypanosoma brucei brucei/enzimologia
20.
J Mass Spectrom ; 42(3): 370-9, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17212372

RESUMO

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.


Assuntos
Carboidratos/química , Proteínas de Membrana/química , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sítios de Ligação , Glicosilação , Humanos , Ligação Proteica
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