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Immunochemotherapy has been the mainstay of treatment for newly diagnosed diffuse large B-cell lymphoma (ndDLBCL) yet is inadequate for many patients. In this work, we perform unsupervised clustering on transcriptomic features from a large cohort of ndDLBCL patients and identify seven clusters, one called A7 with poor prognosis, and develop a classifier to identify these clusters in independent ndDLBCL cohorts. This high-risk cluster is enriched for activated B-cell cell-of-origin, low immune infiltration, high MYC expression, and copy number aberrations. We compare and contrast our methodology with recent DLBCL classifiers to contextualize our clusters and show improved prognostic utility. Finally, using pre-clinical models, we demonstrate a mechanistic rationale for IKZF1/3 degraders such as lenalidomide to overcome the low immune infiltration phenotype of A7 by inducing T-cell trafficking into tumors and upregulating MHC I and II on tumor cells, and demonstrate that TCF4 is an important regulator of MYC-related biology in A7.
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Regulação Neoplásica da Expressão Gênica , Fator de Transcrição Ikaros , Lenalidomida , Linfoma Difuso de Grandes Células B , Proteínas Proto-Oncogênicas c-myc , Fator de Transcrição 4 , Transcriptoma , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Lenalidomida/uso terapêutico , Lenalidomida/farmacologia , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Fator de Transcrição 4/genética , Fator de Transcrição 4/metabolismo , Linfócitos B/metabolismo , Linfócitos B/imunologia , Prognóstico , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Variações do Número de Cópias de DNARESUMO
Demyelination is a common pathological feature in a wide range of diseases, characterized by the loss of myelin sheath and myelin-supporting oligodendrocytes. These losses lead to impaired axonal function, increased vulnerability of axons to damage, and result in significant brain atrophy and neuro-axonal degeneration. Multiple pathomolecular processes contribute to neuroinflammation, oligodendrocyte cell death, and progressive neuronal dysfunction. In this study, we use the cuprizone mouse model of demyelination to investigate long-term non-invasive gamma entrainment using sensory stimulation as a potential therapeutic intervention for promoting myelination and reducing neuroinflammation in male mice. Here, we show that multisensory gamma stimulation mitigates demyelination, promotes oligodendrogenesis, preserves functional integrity and synaptic plasticity, attenuates oligodendrocyte ferroptosis-induced cell death, and reduces brain inflammation. Thus, the protective effects of multisensory gamma stimulation on myelin and anti-neuroinflammatory properties support its potential as a therapeutic approach for demyelinating disorders.
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Cuprizona , Doenças Desmielinizantes , Modelos Animais de Doenças , Bainha de Mielina , Oligodendroglia , Animais , Cuprizona/toxicidade , Masculino , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/terapia , Doenças Desmielinizantes/patologia , Camundongos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Bainha de Mielina/metabolismo , Camundongos Endogâmicos C57BL , Ferroptose , Plasticidade Neuronal , Encéfalo/patologia , Encéfalo/metabolismo , Doenças Neuroinflamatórias/induzido quimicamente , Doenças Neuroinflamatórias/patologiaRESUMO
Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.
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Recent genetic and molecular classification of DLBCL has advanced our knowledge of disease biology, yet were not designed to predict early events and guide anticipatory selection of novel therapies. To address this unmet need, we used an integrative multiomic approach to identify a signature at diagnosis that will identify DLBCL at high risk of early clinical failure. Tumor biopsies from 444 newly diagnosed DLBCL were analyzed by WES and RNAseq. A combination of weighted gene correlation network analysis and differential gene expression analysis was used to identify a signature associated with high risk of early clinical failure independent of IPI and COO. Further analysis revealed the signature was associated with metabolic reprogramming and identified cases with a depleted immune microenvironment. Finally, WES data was integrated into the signature and we found that inclusion of ARID1A mutations resulted in identification of 45% of cases with an early clinical failure which was validated in external DLBCL cohorts. This novel and integrative approach is the first to identify a signature at diagnosis, in a real-world cohort of DLBCL, that identifies patients at high risk for early clinical failure and may have significant implications for design of therapeutic options.
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Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/diagnóstico , Masculino , Feminino , Perfilação da Expressão Gênica , Pessoa de Meia-Idade , Transcriptoma , Mutação , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Biomarcadores Tumorais/genética , Idoso , Prognóstico , Microambiente Tumoral , Sequenciamento do Exoma , Adulto , Proteínas de Ligação a DNA/genética , Falha de TratamentoRESUMO
The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/ß) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.
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Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismoRESUMO
The autosomal recessive disorder Ataxia-Telangiectasia is caused by a dysfunction of the stress response protein, ATM. In the nucleus of proliferating cells, ATM senses DNA double-strand breaks and coordinates their repair. This role explains T-cell dysfunction and tumour risk. However, it remains unclear whether this function is relevant for postmitotic neurons and underlies cerebellar atrophy, since ATM is cytoplasmic in postmitotic neurons. Here, we used ATM-null mice that survived early immune deficits via bone-marrow transplantation, and that reached initial neurodegeneration stages at 12 months of age. Global cerebellar transcriptomics demonstrated that ATM depletion triggered upregulations in most neurotransmission and neuropeptide systems. Downregulated transcripts were found for the ATM interactome component Usp2, many non-coding RNAs, ataxia genes Itpr1, Grid2, immediate early genes and immunity factors. Allelic splice changes affected prominently the neuropeptide machinery, e.g., Oprm1. Validation experiments with stressors were performed in human neuroblastoma cells, where ATM was localised only to cytoplasm, similar to the brain. Effect confirmation in SH-SY5Y cells occurred after ATM depletion and osmotic stress better than nutrient/oxidative stress, but not after ATM kinase inhibition or DNA stressor bleomycin. Overall, we provide pioneer observations from a faithful A-T mouse model, which suggest general changes in synaptic and dense-core vesicle stress adaptation.
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Neuroblastoma , Doenças Neurodegenerativas , Neuropeptídeos , Camundongos , Animais , Humanos , Lactente , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Regulação para Baixo , Regulação para Cima , Transcriptoma/genética , Transmissão Sináptica/genética , Doenças Neurodegenerativas/metabolismo , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , DNA , RNA não Traduzido , Atrofia , Receptores de Inositol 1,4,5-Trifosfato/metabolismoRESUMO
c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.
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Proteínas Tirosina Quinases , Secretoma , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Fosfotransferases , Fosforilação , Domínios de Homologia de src , Comunicação Celular , Quinases da Família srcRESUMO
BACKGROUND: COV-BOOST is a multicentre, randomised, controlled, phase 2 trial of seven COVID-19 vaccines used as a third booster dose in June 2021. Monovalent messenger RNA (mRNA) COVID-19 vaccines were subsequently widely used for the third and fourth-dose vaccination campaigns in high-income countries. Real-world vaccine effectiveness against symptomatic infections following third doses declined during the Omicron wave. This report compares the immunogenicity and kinetics of responses to third doses of vaccines from day (D) 28 to D242 following third doses in seven study arms. METHODS: The trial initially included ten experimental vaccine arms (seven full-dose, three half-dose) delivered at three groups of six sites. Participants in each site group were randomised to three or four experimental vaccines, or MenACWY control. The trial was stratified such that half of participants had previously received two primary doses of ChAdOx1 nCov-19 (Oxford-AstraZeneca; hereafter referred to as ChAd) and half had received two doses of BNT162b2 (Pfizer-BioNtech, hereafter referred to as BNT). The D242 follow-up was done in seven arms (five full-dose, two half-dose). The BNT vaccine was used as the reference as it was the most commonly deployed third-dose vaccine in clinical practice in high-income countries. The primary analysis was conducted using all randomised and baseline seronegative participants who were SARS-CoV-2 naïve during the study and who had not received a further COVID-19 vaccine for any reason since third dose randomisation. RESULTS: Among the 817 participants included in this report, the median age was 72 years (IQR: 55-78) with 50.7% being female. The decay rates of anti-spike IgG between vaccines are different among both populations who received initial doses of ChAd/ChAd and BNT/BNT. In the population that previously received ChAd/ChAd, mRNA vaccines had the highest titre at D242 following their vaccine dose although Ad26. COV2. S (Janssen; hereafter referred to as Ad26) showed slower decay. For people who received BNT/BNT as their initial doses, a slower decay was also seen in the Ad26 and ChAd arms. The anti-spike IgG became significantly higher in the Ad26 arm compared to the BNT arm as early as 3 months following vaccination. Similar decay rates were seen between BNT and half-BNT; the geometric mean ratios ranged from 0.76 to 0.94 at different time points. The difference in decay rates between vaccines was similar for wild-type live virus-neutralising antibodies and that seen for anti-spike IgG. For cellular responses, the persistence was similar between study arms. CONCLUSIONS: Heterologous third doses with viral vector vaccines following two doses of mRNA achieve more durable humoral responses compared with three doses of mRNA vaccines. Lower doses of mRNA vaccines could be considered for future booster campaigns.
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COVID-19 , Vacinas Virais , Feminino , Humanos , Idoso , Masculino , Vacinas contra COVID-19 , Vacina BNT162 , ChAdOx1 nCoV-19 , COVID-19/prevenção & controle , SARS-CoV-2 , Imunidade , Reino Unido , Imunoglobulina G , Anticorpos Antivirais , Vacinação , Imunogenicidade da VacinaRESUMO
Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.
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Neoplasias , Quimera de Direcionamento de Proteólise , Camundongos , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
INTRODUCTION AND IMPORTANCE: Transverse colon volvulus is a rare cause of colonic obstruction accounting for 1-3 % of colonic volvuli due to the short mesentery and hepatic and splenic attachments. Previous mobilisation of the flexures and conditions resulting in chronic dilatation of the colon predispose to the condition. The risk of mortality is high, ranging from 11 to 20 % highlighting the need for early diagnosis and intervention. CASE PRESENTATION: We present a case of a 90-year-old male who was referred with a large bowel obstruction with a transition at the splenic flexure and the liver rotated to the left upper quadrant. The patient was taken to the theatre and an emergency laparotomy was performed with findings of a transverse colon volvulus. The liver was initially found in the left upper quadrant and was freely mobile in the upper abdomen consistent with an absence of the hepatic ligament. A subtotal colectomy was performed. Unfortunately, the postoperative course was complicated by a cardiac event and the patient died on postoperative day six. CLINICAL DISCUSSION: Absence of hepatic ligaments is a rare cause of transverse colon volvulus which has only been described in one previous case report. Diagnosis of transverse colon volvulus can be challenging and early operative intervention with colectomy is required to minimise mortality. CONCLUSION: This case illustrates an unusual cause of transverse colon volvulus secondary to the absence of the hepatic ligaments and stresses the need for early diagnosis and intervention due to the high mortality associated with this condition.
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Millions of traumatic brain injuries (TBIs) occur annually. TBIs commonly result from falls, traffic accidents, and sports-related injuries, all of which involve rotational acceleration/deceleration of the brain. During these injuries, the brain endures a multitude of primary insults including compression of brain tissue, damaged vasculature, and diffuse axonal injury. All of these deleterious effects can contribute to secondary brain ischemia, cellular death, and neuroinflammation that progress for weeks, months, and lifetime after injury. While the linear effects of head trauma have been extensively modeled, less is known about how rotational injuries mediate neuronal damage following injury. Here, we developed a new model of repetitive rotational head trauma in rodents and demonstrated acute and prolonged pathological, behavioral, and electrophysiological effects of rotational TBI (rTBI). We identify aberrant Cyclin-dependent kinase 5 (Cdk5) activity as a principal mediator of rTBI. We utilized Cdk5-enriched phosphoproteomics to uncover potential downstream mediators of rTBI and show pharmacological inhibition of Cdk5 reduces the cognitive and pathological consequences of injury. These studies contribute meaningfully to our understanding of the mechanisms of rTBI and how they may be effectively treated.
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Lesões Encefálicas Traumáticas , Traumatismos Craniocerebrais , Quinase 5 Dependente de Ciclina , Animais , Ratos , Encéfalo , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/metabolismo , Traumatismos Craniocerebrais/genética , Traumatismos Craniocerebrais/metabolismo , Quinase 5 Dependente de Ciclina/metabolismoRESUMO
Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.
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Aminoacil-tRNA Sintetases , Mieloma Múltiplo , Humanos , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismoRESUMO
Introduction: Traumatic injuries from jet ski-related accidents have increased in incidence over the past few decades. Anorectal injuries are uncommon but typically arise from high-speed jet ski accidents. We present a case of a severe anorectal injury from a fall off the back of an accelerating jet ski. Case report: This case reports on the presentation, operative findings and management of a 22-year-old female with major internal and external anal sphincter disruption sustained via an unusual traumatic mechanism. Operative findings identified a complete internal and external anal sphincter disruption at the 1 and 7 O'clock positions and extra-peritoneal rectal perforation. Washout, suture repair and an end-colostomy were performed. Conclusion: Understanding the potential severity of injury from the insult mechanism is paramount to triaging and managing trauma patients. Although this case describes an inconspicuous mechanism, the resulting trauma is significant and should prompt consideration in future cases. In addition, the article describes an approach to the repair of such injuries and the difficult decision relating to the role and type of defunctioning colostomy to protect any possible missed injuries in a complex traumatic environment, and in the protection of the anorectal repair.
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Immunomodulatory drugs (IMiDs), including lenalidomide and pomalidomide, are used in the routine treatment for multiple myeloma (MM) patients. Cereblon (CRBN) is the direct molecular target of IMiDs. While CRBN is not an essential gene for MM cell proliferation, the frequency of CRBN genetic aberrations, including mutation, copy number loss, and exon-10 (which includes a portion of the IMiD-binding domain) splicing, have been reported to incrementally increase in later-line patients. CRBN exon-10 splicing has also been shown to be associated with decreased progression-free survival in both newly diagnosed and relapsed refractory MM patients. Although we did not find significant general splicing defects among patients with CRBN exon-10 splice variant when compared to those expressing the full-length transcript, we identified upregulated TNFA signaling via NFKB, inflammatory response, and IL-10 signaling pathways in patients with exon-10 splice variant across various data sets-all potentially promoting tumor growth via chronic growth signals. We examined master regulators that mediate transcriptional programs in CRBN exon-10 splice variant patients and identified BATF, EZH2, and IKZF1 as the key candidates across the four data sets. Upregulated downstream targets of BATF, EZH2, and IKZF1 are components of TNFA signaling via NFKB, IL2/STAT5 signaling pathways, and IFNG response pathways. Previously, BATF-mediated transcriptional regulation was associated with venetoclax sensitivity in MM. Interestingly, we found that an EZH2 sensitivity gene expression signature also correlated with high BATF or venetoclax sensitivity scores in these tumors. Together, these data provide a rationale for investigating EZH2 inhibitors or venetoclax in combination with the next generation CRBN-targeting agents, such as CELMoDs, for patients overexpressing the CRBN exon-10 splice variant.
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AIM: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. METHODS: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30 min/48 hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . RESULTS: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1ß and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. CONCLUSION: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.
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Injúria Renal Aguda , Fosfatase 3 de Especificidade Dupla , Traumatismo por Reperfusão , Injúria Renal Aguda/metabolismo , Animais , Fosfatase 3 de Especificidade Dupla/genética , Células Endoteliais/metabolismo , Inflamação/genética , Inflamação/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismoRESUMO
Ubiquitination is a post-translational modification that affects protein degradation as well as a variety of cellular processes. Methods that globally profile ubiquitination are powerful tools to better understand these processes. Here we describe an updated method for identification and quantification of thousands of sites of ubiquitination from cells, tissues, or other biological materials. The method involves cell lysis and digestion to peptides, immunoaffinity enrichment with an antibody recognizing di-glycine remnants left behind at ubiquitinated lysines, and liquid chromatography-tandem mass spectrometry analysis of the enriched peptides.