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1.
J Pharm Biomed Anal ; 209: 114537, 2022 Feb 05.
Article in English | MEDLINE | ID: mdl-34929569

ABSTRACT

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).


Subject(s)
Erwinia , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Asparaginase , Chromatography, Liquid , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Tandem Mass Spectrometry
2.
Toxicol In Vitro ; 62: 104697, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31669365

ABSTRACT

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.


Subject(s)
Dermatitis, Allergic Contact/pathology , Haptens/chemistry , Immunization , Proteins/chemistry , Proteomics/methods , Skin/drug effects , Acrylates/toxicity , Cell Line , Cyclopropanes/toxicity , Dermatitis, Allergic Contact/immunology , Humans , Mass Spectrometry , Models, Molecular , Serum Albumin/chemistry
3.
Cancer Cell Int ; 18: 71, 2018.
Article in English | MEDLINE | ID: mdl-29760584

ABSTRACT

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.
Article in English | MEDLINE | ID: mdl-29154981

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/therapeutic use , Erlotinib Hydrochloride/therapeutic use , Lapatinib/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Receptor, ErbB-2/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/physiology , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/physiology , Humans , Signal Transduction , Urinary Bladder Neoplasms/etiology
5.
Cell Signal ; 28(4): 284-93, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26795954

ABSTRACT

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.


Subject(s)
Autophagy , Elongation Factor 2 Kinase/metabolism , Fibroblasts/enzymology , Protein Biosynthesis/physiology , Animals , Cell Survival , Elongation Factor 2 Kinase/genetics , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
6.
Mol Cell Biol ; 35(10): 1788-804, 2015 May.
Article in English | MEDLINE | ID: mdl-25755286

ABSTRACT

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.


Subject(s)
Cell Hypoxia , Elongation Factor 2 Kinase/metabolism , Neurons/enzymology , Proline/metabolism , Animals , Calmodulin/metabolism , Catalytic Domain , Cells, Cultured , Elongation Factor 2 Kinase/chemistry , Enzyme Activation , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Hydroxylation , Mice , Peptide Elongation Factor 2/metabolism , Phosphorylation/drug effects , Prolyl Hydroxylases/metabolism , Prolyl-Hydroxylase Inhibitors/pharmacology
7.
Mol Cell Biol ; 34(22): 4088-103, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25182533

ABSTRACT

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.


Subject(s)
Elongation Factor 2 Kinase/metabolism , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Animals , Cell Line , Cell Line, Tumor , Elongation Factor 2 Kinase/genetics , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , MAP Kinase Signaling System , Mechanistic Target of Rapamycin Complex 1 , Mice , Neoplasms/genetics , Phosphorylation , Point Mutation , Rats , raf Kinases/metabolism , ras Proteins/metabolism
8.
Mol Cell Biol ; 34(12): 2294-307, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24732796

ABSTRACT

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.


Subject(s)
Catalytic Domain , Conserved Sequence , Elongation Factor 2 Kinase/chemistry , Elongation Factor 2 Kinase/metabolism , Amino Acid Sequence , Amino Acids/genetics , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation , Phosphothreonine/metabolism , Protein Binding , Protein Structure, Secondary , Protozoan Proteins/chemistry , Structural Homology, Protein , Structure-Activity Relationship , Substrate Specificity
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