Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA.

The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.

RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements.

Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization.

Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.

Oncogenic RET kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans.

To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation.

Regulation of the localisation and function of the oncogene LYRIC/AEG-1 by ubiquitination at K486 and K491.

The pivotal role of LYRIC/AEG-1 in malignant transformation, tumourigenesis and chemo-resistance has previously been demonstrated in different cell types and sub-cellular compartments. The localisation of LYRIC/AEG-1 appears crucial to its function and is regulated by three lysine-rich nuclear localisation signal regions, one of which was previously demonstrated to be modified by ubiquitin. Here we show that mutation of LYRIC/AEG-1 at K486 and K491 results in a loss of ubiquitination. A K486/491R double mutant that is incapable of ubiquitination shows reduced binding to the NFκB subunit p65 or importin-ß resulting in a distinctive peri-nuclear localisation of LYRIC/AEG-1. We also provide evidence to suggest that TOPORS, an E3 ligase that also regulates p53 modification may be responsible for LYRIC/AEG-1 ubiquitin modification. Overall we demonstrate that specific sites of LYRIC/AEG-1 ubiquitination are essential for regulating LYRIC/AEG-1 localisation and functionally interacting proteins.

Binding of dynein intermediate chain 2 to paxillin controls focal adhesion dynamics and migration.

In migrating NRK cells, aPKCs control the dynamics of turnover of paxillin-containing focal adhesions (FA) determining migration rate. Using a proteomic approach (two-dimensional fluorescence difference gel electrophoresis), dynein intermediate chain 2 (dynein IC2) was identified as a protein that is phosphorylated inducibly during cell migration in a PKC-regulated manner. By gene silencing and co-immunoprecipitation studies, we show that dynein IC2 regulates the speed of cell migration through its interaction with paxillin. This interaction is controlled by serine 84 phosphorylation, which lies on the aPKC pathway. The evidence presented thus links aPKC control of migration to the dynein control of FA turnover through paxillin.

Differential protein expression, DNA binding and interaction with SV40 large tumour antigen implicate the p63-family of proteins in replicative senescence.

P53 activity plays a key role in mammalian cells when they undergo replicative senescence at their Hayflick limit. To determine whether p63 proteins, members of the family of p53-related genes, are also involved in this process, we examined their expression in serially passaged rat embryo fibroblasts. Upon senescence, two truncated DeltaNp63 proteins decreased in abundance whereas two TAp63 isoforms accumulated. 2-D gel analysis showed that the DeltaNp63 proteins underwent post-translational modifications in both proliferating and senescent cells. Direct binding of DeltaNp63 proteins to a p53 consensus motif was greater in proliferating cells than senescent cells. In contrast p63alpha isoforms bound to DNA in a p53 dependent manner and this was higher in senescent cells than proliferating cells. An interaction of p63alpha proteins with SV40 large tumour antigen was also detected and ectopic expression of DeltaNp63alpha can extend the lifespan of rat embryo fibroblasts. Taken together the results indicate that p63 proteins may play a role in replicative senescence either by competition for p53 DNA binding sites or by direct interaction with p53 protein bound to DNA.

H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression.

To defend against the potential damages induced by reactive oxygen species, proliferating cells enter a transient cell cycle arrest. We treated mouse fibroblasts with H(2)O(2) and found that sublethal doses of H(2)O(2) induced a transient multi-phase cell cycle arrest at the G(1), S, and G(2) phases but not the M phase. Western blot analysis demonstrated that this transient cell cycle arrest is associated with the down-regulation of cyclins D1 and D3 and up-regulation of the CKI p21(Cip1) expression. We also demonstrate that the induction in p21(Cip1) expression by H(2)O(2) is at least partially mediated at the transcriptional level and can occur in the absence of p53 function. Further immunoprecipitation kinase and immunodepletion assays indicated that in response to H(2)O(2) treatment, the down-regulation of cyclin Ds expression are associated with repression of cyclin D-CDK4, whereas the accumulation of p21(Cip1) is responsible for the inhibition of cyclin E and A-CDK2 activity and associated with the down-regulation of cyclin B-CDC2 activity. These data could account for the cell cycle arrest at the G(1), S, and G(2) phases following H(2)O(2) stimulation. Deletion of p21(Cip1), restoration of cyclin D expression, or overexpression of cyclin E alone is insufficient to effectively overcome the cell cycle arrest caused by sublethal doses of H(2)O(2). By contrast, overexpression of the human Herpesvirus 8 K cyclin, which can mimic the function of cyclin D and E, is enough to override this transient cell cycle arrest. On the basis of our findings, we propose a model in which moderate levels of H(2)O(2) induce a transient multi-phase cell cycle arrest at least partially through up-regulation of p21(Cip1) and down-regulation of cyclin D expression.