Evolutionary and cellular analysis of the 'dark' pseudokinase PSKH2.

Pseudokinases, so named because they lack one or more conserved canonical amino acids that define their catalytically active relatives, have evolved a variety of biological functions in both prokaryotic and eukaryotic organisms. Human PSKH2 is closely related to the canonical kinase PSKH1, which maps to the CAMK family of protein kinases. Primates encode PSKH2 in the form of a pseudokinase, which is predicted to be catalytically inactive due to loss of the invariant catalytic Asp residue. Although the biological role(s) of vertebrate PSKH2 proteins remains unclear, we previously identified species-level adaptions in PSKH2 that have led to the appearance of kinase or pseudokinase variants in vertebrate genomes alongside a canonical PSKH1 paralog. In this paper we confirm that, as predicted, PSKH2 lacks detectable protein phosphotransferase activity, and exploit structural informatics, biochemistry and cellular proteomics to begin to characterise vertebrate PSKH2 orthologues. AlphaFold 2-based structural analysis predicts functional roles for both the PSKH2 N- and C-regions that flank the pseudokinase domain core, and cellular truncation analysis confirms that the N-terminal domain, which contains a conserved myristoylation site, is required for both stable human PSKH2 expression and localisation to a membrane-rich subcellular fraction containing mitochondrial proteins. Using mass spectrometry-based proteomics, we confirm that human PSKH2 is part of a cellular mitochondrial protein network, and that its expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system. HSP90 interactions are mediated through binding to the PSKH2 C-terminal tail, leading us to predict that this region might act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signalling.

Pharmacological impact of FLT3 mutations on receptor activity and responsiveness to tyrosine kinase inhibitors.

Acute myelogenous leukaemia (AML) is an aggressive blood cancer characterized by the rapid proliferation of immature myeloid blast cells, resulting in a high mortality rate. The 5-year overall survival rate for AML patients is approximately 25%. Circa 35% of all patients carry a mutation in the FLT3 gene which have a poor prognosis. Targeting FLT3 receptor tyrosine kinase has become a treatment strategy in AML patients possessing FLT3 mutations. The most common mutations are internal tandem duplications (ITD) within exon 14 and a single nucleotide polymorphism (SNP) that leads to a point mutation in the D835 of the tyrosine kinase domain (TKD). Variations in the ITD sequence and the occurrence of other point mutations that lead to ligand-independent FLT3 receptor activation create difficulties in developing personalized therapeutic strategies to overcome observed mutation-driven drug resistance. Midostaurin and quizartinib are tyrosine kinase inhibitors (TKIs) with inhibitory efficacy against FLT3-ITD, but exhibit limited clinical impact. In this review, we focus on the structural aspects of the FLT3 receptor and correlate those mutations with receptor activation and the consequences for molecular and clinical responsiveness towards therapies targeting FLT3-ITD positive AML.

Determining site occupancy of acetaminophen covalent binding to target proteins in vitro.

Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.

Polymorphic variants of MRP4/ABCC4 differentially modulate the transport of methylated arsenic metabolites and physiological organic anions.

Broad inter-individual variation exists in susceptibility to arsenic-induced tumours, likely involving differences in the ability of individuals to eliminate this metalloid. We recently identified human multidrug resistance protein 4 (MRP4/ABCC4) as a novel pathway for the cellular export of dimethylarsinic acid (DMAV), the major urinary arsenic metabolite in humans, and the diglutathione conjugate of the highly toxic monomethylarsonous acid [MMA(GS)2]. These findings, together with the basolateral and apical membrane localization of MRP4 in hepatocytes and renal proximal tubule cells, respectively, suggest a role for MRP4 in the urinary elimination of hepatic arsenic metabolites. Accordingly, we have now investigated the influence of non-synonymous single nucleotide polymorphisms (SNPs) on MRP4 levels, cellular localization, and arsenical transport. Of eight MRP4 variants (C171G-, G187W-, K304N-, G487E-, Y556C-, E757K-, V776I- and C956S-MRP4) characterized, two (V776I- and C956S-MRP4) did not localize appropriately to the plasma membrane of HEK293T and LLC-PK1 cells. Characterization of the six correctly localized mutants revealed that MMA(GS)2 transport by C171G-, G187W-, and K304N-MRP4 was 180%, 73%, and 30% of WT-MRP4 activity, respectively, whereas DMAV transport by K304N- and Y556C-MRP4 was 30% and 184% of WT-MRP4, respectively. Transport of the prototypical physiological MRP4 substrates prostaglandin E2 and 17ß-estradiol 17-(ß-d-glucuronide) by the six variants was also differentially affected. Thus, MRP4 variants have differing abilities to transport arsenic and endogenous metabolites through both altered function and membrane localization. Further investigation is warranted to determine if genetic variations in ABCC4 contribute to inter-individual differences in susceptibility to arsenic-induced (and potentially other) diseases.

Arsenic Triglutathione [As(GS)3] Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) Is Selectively Modified by Phosphorylation of Tyr920/Ser921 and Glycosylation of Asn19/Asn23.

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) is responsible for the cellular export of a chemically diverse array of xenobiotics and endogenous compounds. Arsenic, a human carcinogen, is a high-affinity MRP1 substrate as arsenic triglutathione [As(GS)3]. In this study, marked differences in As(GS)3 transport kinetics were observed between MRP1-enriched membrane vesicles prepared from human embryonic kidney 293 (HEK) (Km 3.8 µM and Vmax 307 pmol/mg per minute) and HeLa (Km 0.32 µM and Vmax 42 pmol/mg per minute) cells. Mutant MRP1 lacking N-linked glycosylation [Asn19/23/1006Gln; sugar-free (SF)-MRP1] expressed in either HEK293 or HeLa cells had low Km and Vmax values for As(GS)3, similar to HeLa wild-type (WT) MRP1. When prepared in the presence of phosphatase inhibitors, both WT- and SF-MRP1-enriched membrane vesicles had a high Km value for As(GS)3 (3-6 µM), regardless of the cell line. Kinetic parameters of As(GS)3 for HEK-Asn19/23Gln-MRP1 were similar to those of HeLa/HEK-SF-MRP1 and HeLa-WT-MRP1, whereas those of single glycosylation mutants were like those of HEK-WT-MRP1. Mutation of 19 potential MRP1 phosphorylation sites revealed that HEK-Tyr920Phe/Ser921Ala-MRP1 transported As(GS)3 like HeLa-WT-MRP1, whereas individual HEK-Tyr920Phe- and -Ser921Ala-MRP1 mutants were similar to HEK-WT-MRP1. Together, these results suggest that Asn19/Asn23 glycosylation and Tyr920/Ser921 phosphorylation are responsible for altering the kinetics of MRP1-mediated As(GS)3 transport. The kinetics of As(GS)3 transport by HEK-Asn19/23Gln/Tyr920Glu/Ser921Glu were similar to HEK-WT-MRP1, indicating that the phosphorylation-mimicking substitutions abrogated the influence of Asn19/23Gln glycosylation. Overall, these data suggest that cross-talk between MRP1 glycosylation and phosphorylation occurs and that phosphorylation of Tyr920 and Ser921 can switch MRP1 to a lower-affinity, higher-capacity As(GS)3 transporter, allowing arsenic detoxification over a broad concentration range.

Ethnopharmacognostic survey on botanical compendia for potential cosmeceutic species from Atlantic Forest

The Atlantic Forest is one of the most endangered ecosystems on earth, and is acknowledged as an area with truly exceptional levels of biodiversity under enormous levels of stress. Cosmeceutics cover a border area between pharmaceuticals for skin diseases and cosmetics. Natural products for external application, to improve the appearance of the skin or for skin treatment, have always been observed and used by native cultures. The present work deals with the ethnopharmacognostic analysis of two botanical compendia (BC), named: Dicionário das Plantas Úteis do Brasil - e das exóticas cultivadas, compiled by Pio Correa (PC) Flora Ilustrada Catarinense (FIC). From these BC, reported species with cosmeceutical uses or with related physico-chemical or organoleptic characteristics were selected, updated, searched for scientific background and highlighted if endangered. PC and FIC specified that 245 plant species, belonging to 98 plant families, are used in Brazil for cosmeceutical, cosmetic or skin remedies. The families most widely represented were Asteraceae, Fabaceae, Myrtaceae, Annonaceae, Clusiaceae, Anacardiaceae, Apiaceae, Bignoniaceae and Solanaceae The most frequently cited plant parts were bark, followed by leaves and aerial parts. The most frequently cited properties were astringency and tonic effect followed by uses in skin disorders and wound healing, emollient characteristic, anti-inflammatory uses and healing of skin ulcers, antiseptic effects, parasiticide and skin lightening properties and aphrodisiacs. According to the Pubmed survey, most of the selected species (65 percent) have not been previously investigated for potential cosmeceutical applications, nor have their chemical composition been investigated.

A Mata Atlântica é um dos ecossistemas mais ameaçados do planeta, sendo reconhecida como uma área de grande biodiversidade sob alto nível de stress. A área cosmecêutica abrange medicamentos de uso tópico e cosméticos, e o uso de produtos naturais para aplicação externa sempre foi observado em diversas culturas. Este trabalho trata de uma análise etnofarmacognóstica de dois compêndios botânicos (CB): Dicionário das Plantas Úteis do Brasil - e das exóticas cultivadas, compilado por Pio Correa (PC) e Flora Ilustrada Catarinense (FIC). Destes compêndios, foram selecionadas espécies com uso cosmecêutico ou com características fisico-químicas e organolépticas relacionadas. Essas espécies selecionadas foram analisadas quanto à validade da nomenclatura botânica e a ocorrência de publicação científica, e quanto ao risco de extinção. PC e FIC apontaram que 245 espécies vegetais, pertencendo a 98 famílias, possuem uso cosmecêutico no Brasil. As famílias mais citadas foram: Asteraceae, Fabaceae, Myrtaceae, Annonaceae, Clusiaceae, Anacardiaceae, Apiaceae, Bignoniaceae e Solanaceae. As partes usadas mais citadas foram cascas, folhas e partes aéreas. As propriedades mais citadas foram efeito tônico e adstringente, seguido de efeito cicatrizante, emoliente, antiinflamatório, antiúlcera, anti-séptico, parasiticida e clareador da pele. De acordo com a pesquisa bibliográfica no Pubmed, a maioria das espécies selecionadas (65 por cento) não foi investigada farmacológica e quimicamente.