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1.
J Pharm Biomed Anal ; 209: 114537, 2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-34929569

RESUMO

Erwinase® or Erwinaze® are the proprietary names for the L-asparaginase enzyme derived from Erwinia chrysanthemi.L-asparaginase is an integral part of the treatment of Acute Lymphoblastic Leukaemia (ALL) in children and adolescents. E. chrysanthemiL-asparaginase was first developed in the early 1970s at Porton Down and is currently manufactured by Porton Biopharma Ltd. One of the early purification steps during E. chrysanthemiL-asparaginase manufacture, involves use of batch cation exchange carboxymethyl resin, and alternatives to this older technology are currently under investigation using mass spectrometry to understand the impact of resin changes on the impurity profile. In this study, a novel SWATH library was developed for E. chrysanthemi proteome and used to evaluate this potential process change on product yield and host cell protein (HCP) profile and clearance. An ELISA assay is currently used as a quality control release test for quantifying HCPs at the Drug Substance (DS) stage, but these early extract samples are too crude for interference-free analysis by ELISA. Given that ELISA assay could not be used in the assessment of new resin options, SWATH LC-MS/MS analysis proved to be pivotal in selecting a resin for further scale-up and implementation. The data quantified that L-asparaginase from the new process step was 2.28-fold higher in concentration than in legacy-process samples. The new step, using a modern ion exchanger, was at least equivalent and in some cases outperformed the legacy resin step in terms of HCP clearance for 78.2% of total HCPs (528 of 675 total proteins).


Assuntos
Erwinia , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Asparaginase , Cromatografia Líquida , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Espectrometria de Massas em Tandem
2.
Toxicol In Vitro ; 62: 104697, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31669365

RESUMO

The potential risk of skin sensitisation, associated with the development of allergic contact dermatitis (ACD), is a consideration in the safety assessment of new ingredients for use in personal care products. Protein haptenation in skin by sensitising chemicals is the molecular initiating event causative of skin sensitisation. Current methods for monitoring skin sensitisation rely on limited reactivity assays, motivating interest in the development of proteomic approaches to characterise the skin haptenome. Increasing our mechanistic understanding of skin sensitisation and ACD using proteomics presents an opportunity to develop non-animal predictive methods and/or risk assessment approaches. Previously, we have used a novel stable isotope labelling approach combined with data independent mass spectrometry (HDMSE) to characterise the haptenome for a number of well-known sensitisers. We have now extended this work by characterising the haptenome of the sensitisers Diphenylcyclopropenone (DPCP) and Ethyl Acrylate (EA) with the model protein Human Serum Albumin (HSA) and the complex lysates of the skin keratinocyte, HaCaT cell line. We show that haptenation in complex nucleophilic models is not random, but a specific, low level and reproducible event. Proteomic analysis extends our understanding of sensitiser reactivity beyond simple reactivity assays and offers a route to monitoring haptenation in living cells.


Assuntos
Dermatite Alérgica de Contato/patologia , Haptenos/química , Imunização , Proteínas/química , Proteômica/métodos , Pele/efeitos dos fármacos , Acrilatos/toxicidade , Linhagem Celular , Ciclopropanos/toxicidade , Dermatite Alérgica de Contato/imunologia , Humanos , Espectrometria de Massas , Modelos Moleculares , Albumina Sérica/química
3.
Cancer Cell Int ; 18: 71, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29760584

RESUMO

BACKGROUND: Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7. METHODS: We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7. RESULTS: Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments. CONCLUSION: LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted.

4.
Urology ; 112: 225.e1-225.e7, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29154981

RESUMO

OBJECTIVE: To investigate perturbations in downstream signaling pathway activation and potential resistance mechanisms to epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) inhibition in cell line models of bladder cancer. METHODS: We undertook a structured screening approach by phosphokinase array, followed by validation steps, to detect activated downstream signaling pathway nodes after therapeutic inhibition of EGFR or HER2 in bladder cancer cell lines. RESULTS: Erlotinib treatment of RT112 cells induced phosphorylation of 9 activated phosphoprotein targets (p38 mitogen-activated protein kinase [MAPK] [Thr180/Tyr182], GSK-3α/ß [Ser21/9], MEK1/2 [Ser218/222, Ser222/226], Akt (protein kinase B) [Ser473], TOR [target of rapamycin] [Ser2448], Src [Tyr419], p27 [Thr198], p27 [Thr157], and PLCγ-1 [Tyr783]), whereas STAT4 (signal transducer and activator of transcription 4) (Tyr693) phosphorylation was reduced. Of these, p38 MAPK phosphorylation was confirmed to occur in response to inhibition of either EGFR or HER2 signaling through multiple validation steps, including differing bladder cancer cell lines (RT112, UM-UC-3, and T24) and methods of receptor pathway inhibition (erlotinib, lapatinib, and siRNA depletion of EGFR or HER2). Chemical inhibition of p38 MAPK with SB203580 led to inhibition of proliferation in RT112, UM-UC-3, and T24 cell lines (IC50 20.85, 76.78, and 79.12 µM, respectively). Fractional effect analyses indicated a synergistic interaction for inhibition of cell proliferation when combining SB203580 with lapatinib. CONCLUSION: p38 MAPK is a potential therapeutic target in bladder cancer and this strategy warrants further development in this disease. It may also allow combination therapy strategies to be developed in conjunction with EGFR or HER2 inhibition.


Assuntos
Antineoplásicos/uso terapêutico , Cloridrato de Erlotinib/uso terapêutico , Lapatinib/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Humanos , Transdução de Sinais , Neoplasias da Bexiga Urinária/etiologia
5.
Cell Signal ; 28(4): 284-93, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26795954

RESUMO

Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy.


Assuntos
Autofagia , Quinase do Fator 2 de Elongação/metabolismo , Fibroblastos/enzimologia , Biossíntese de Proteínas/fisiologia , Animais , Sobrevivência Celular , Quinase do Fator 2 de Elongação/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
6.
Mol Cell Biol ; 35(10): 1788-804, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25755286

RESUMO

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.


Assuntos
Hipóxia Celular , Quinase do Fator 2 de Elongação/metabolismo , Neurônios/enzimologia , Prolina/metabolismo , Animais , Calmodulina/metabolismo , Domínio Catalítico , Células Cultivadas , Quinase do Fator 2 de Elongação/química , Ativação Enzimática , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Hidroxilação , Camundongos , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Prolil Hidroxilases/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia
7.
Mol Cell Biol ; 34(22): 4088-103, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25182533

RESUMO

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.


Assuntos
Quinase do Fator 2 de Elongação/metabolismo , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Quinase do Fator 2 de Elongação/genética , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Neoplasias/genética , Fosforilação , Mutação Puntual , Ratos , Quinases raf/metabolismo , Proteínas ras/metabolismo
8.
Mol Cell Biol ; 34(12): 2294-307, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24732796

RESUMO

Eukaryotic elongation factor 2 kinase (eEF2K) is the best-characterized member of the α-kinase family. Within this group, only eEF2K and myosin heavy chain kinases (MHCKs) have known substrates. Here we have studied the roles of specific residues, selected on the basis of structural data for MHCK A and TRPM7, in the function of eEF2K. Our data provide the first information regarding the basis of the substrate specificity of α-kinases, in particular the roles of residues in the so-called N/D loop, which appears to occupy a position in the structure of α-kinases similar to that of the activation loop in other kinases. Several mutations in the EEF2K gene occur in tumors, one of which (Arg303Cys) is at a highly conserved residue in the N/D loop. This mutation greatly enhances eEF2K activity and may be cytoprotective. Our data support the concept that the major autophosphorylation site (Thr348 in eEF2K) docks into a binding pocket to help create the kinase-competent conformation. This is similar to the situation for MHCK A and is consistent with this being a common feature of α-kinases.


Assuntos
Domínio Catalítico , Sequência Conservada , Quinase do Fator 2 de Elongação/química , Quinase do Fator 2 de Elongação/metabolismo , Sequência de Aminoácidos , Aminoácidos/genética , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
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