Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

KIT/PDGFRA inhibitors for the treatment of gastrointestinal stromal tumors: getting to the gist of the problem.

INTRODUCTION: Approximately 90% of gastrointestinal stromal tumors (GISTs) are driven by activating mutations in receptor tyrosine-kinases KIT or PDGFRA. Despite the outstanding results of first-line imatinib in advanced GIST, resistance ultimately occurs mainly through secondary mutations in KIT/PDGFRA. Other tyrosine-kinase inhibitors (TKIs) with a broader spectrum of activity against these mutations are approved after imatinib failure. However, response rates and progression-free survival are drastically lower compared to imatinib. Notably, imatinib also triggers early tolerance adaptation mechanisms, which precede the occurrence of secondary mutations. AREAS COVERED: In this review, we outline the current landscape of KIT inhibitors, discuss the novel agents, and present additional biological pathways that may be therapeutically exploitable. EXPERT OPINION: The development of broad-spectrum and highly selective TKIs able to induce a sustained KIT/PDGFRA inhibition is the pillar of preclinical and clinical investigation in GIST. However, it is now recognized that the situation is more intricate, with various factors interacting with KIT and PDGFRA, playing a crucial role in the response and resistance to treatments. Future strategies in the management of advanced GIST should integrate driver inhibition with the blockade of other molecules to enhance cell death and establish enduring responses in patients.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

The protein phosphatase EYA4 promotes homologous recombination (HR) through dephosphorylation of tyrosine 315 on RAD51.

Efficient DNA repair and limitation of genome rearrangements rely on crosstalk between different DNA double-strand break (DSB) repair pathways, and their synchronization with the cell cycle. The selection, timing and efficacy of DSB repair pathways are influenced by post-translational modifications of histones and DNA damage repair (DDR) proteins, such as phosphorylation. While the importance of kinases and serine/threonine phosphatases in DDR have been extensively studied, the role of tyrosine phosphatases in DNA repair remains poorly understood. In this study, we have identified EYA4 as the protein phosphatase that dephosphorylates RAD51 on residue Tyr315. Through its Tyr phosphatase activity, EYA4 regulates RAD51 localization, presynaptic filament formation, foci formation, and activity. Thus, it is essential for homologous recombination (HR) at DSBs. DNA binding stimulates EYA4 phosphatase activity. Depletion of EYA4 decreases single-stranded DNA accumulation following DNA damage and impairs HR, while overexpression of EYA4 in cells promotes dephosphorylation and stabilization of RAD51, and thereby nucleoprotein filament formation. Our data have implications for a pathological version of RAD51 in EYA4-overexpressing cancers.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Structural Analysis of Janus Tyrosine Kinase Variants in Hematological Malignancies: Implications for Drug Development and Opportunities for Novel Therapeutic Strategies.

Janus tyrosine kinase (JAK) variants are known drivers for hematological disorders. With the full-length structure of mouse JAK1 being recently resolved, new observations on the localization of variants within closed, open, and dimerized JAK structures are possible. Full-length homology models of human wild-type JAK family members were developed using the Glassman et al. reported mouse JAK1 containing the V658F structure as a template. Many mutational sites related to proliferative hematological disorders reside in the JH2 pseudokinase domains facing the region important in dimerization of JAKs in both closed and open states. More than half of all JAK gain of function (GoF) variants are changes in polarity, while only 1.2% are associated with a change in charge. Within a JAK1-JAK3 homodimer model, IFNLR1 (PDB ID7T6F) and the IL-2 common gamma chain subunit (IL2Rγc) were aligned with the respective dimer implementing SWISS-MODEL coupled with ChimeraX. JAK3 variants were observed to encircle the catalytic site of the kinase domain, while mutations in the pseudokinase domain align along the JAK-JAK dimerization axis. FERM domains of JAK1 and JAK3 are identified as a hot spot for hematologic malignancies. Herein, we propose new allosteric surfaces for targeting hyperactive JAK dimers.

Engineering Escherichia coli to produce aromatic chemicals from ethylene glycol.

Microbial overproduction of aromatic chemicals has gained considerable industrial interest and various metabolic engineering approaches have been employed in recent years to address the associated challenges. So far, most studies have used sugars (mostly glucose) or glycerol as the primary carbon source. In this study, we used ethylene glycol (EG) as the main carbon substrate. EG could be obtained from the degradation of plastic and cellulosic wastes. As a proof of concept, Escherichia coli was engineered to transform EG into L-tyrosine, a valuable aromatic amino acid. Under the best fermentation condition, the strain produced 2 g/L L-tyrosine from 10 g/L EG, outperforming glucose (the most common sugar feedstock) in the same experimental conditions. To prove the concept that EG can be converted into different aromatic chemicals, E. coli was further engineered with a similar approach to synthesize other valuable aromatic chemicals, L-phenylalanine and p-coumaric acid. Finally, waste polyethylene terephthalate (PET) bottles were degraded using acid hydrolysis and the resulting monomer EG was transformed into L-tyrosine using the engineered E. coli, yielding a comparable titer to that obtained using commercial EG. The strains developed in this study should be valuable to the community for producing valuable aromatics from EG.

Promoting the activity of a receptor tyrosine phosphatase with a novel pH-responsive transmembrane agonist inhibits cancer-associated phenotypes.

Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal-initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK's phosphorylation and downstream signaling, and ultimately antagonizes RTK-driven cell phenotypes. Here, we designed and tested a series of first-in-class pH-responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR's phosphorylation and inhibited cancer cell EGFR-driven migration and proliferation, similar to the PTPRJ's TM point mutations. Developing tumor-selective and TM-targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP's selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK-driven cancers.

A Single DNA Point Mutation Leads to the Formation of a Cysteine-Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis.

Cysteine dioxygenase (CDO) is a non-heme iron-containing enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C-Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C-Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively non-crosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C-Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

A novel tyrosine tRNA-derived fragment, tRFTyr, induces oncogenesis and lactate accumulation in LSCC by interacting with LDHA.

BACKGROUND: Transfer (t)RNA-derived small RNA (tsRNA), generated from precursor or mature tRNA, is a new type of small non-coding RNA (sncRNA) that has recently been shown to play a vital role in human cancers. However, its role in laryngeal squamous cell carcinoma (LSCC) remains unclear. METHODS: We elucidated the expression profiles of tsRNAs in four paired LSCC and non-neoplastic tissues by sequencing and verified the sequencing data by quantitative real-time PCR (qRT-PCR) of 60 paired samples. The tyrosine-tRNA derivative tRFTyr was identified as a novel oncogene in LSCC for further study. Loss-of-function experiments were performed to evaluate the roles of tRFTyr in tumorigenesis of LSCC. Mechanistic experiments including RNA pull-down, parallel reaction monitoring (PRM) and RNA immunoprecipitation (RIP) were employed to uncover the regulatory mechanism of tRFTyr in LSCC. RESULTS: tRFTyr was significantly upregulated in LSCC samples. Functional assays showed that knockdown of tRFTyr significantly suppressed the progression of LSCC. A series of mechanistic studies revealed that tRFTyr could enhance the phosphorylated level of lactate dehydrogenase A (LDHA) by interacting with it. The activity of LDHA was also activated, which induced lactate accumulation in LSCC cells. CONCLUSIONS: Our data delineated the landscape of tsRNAs in LSCC and identified the oncogenic role of tRFTyr in LSCC. tRFTyr could promote lactate accumulation and tumour progression in LSCC by binding to LDHA. These findings may aid in the development of new diagnostic biomarkers and provide new insights into therapeutic strategies for LSCC.

Predictive data-driven modeling of C-terminal tyrosine function in the EGFR signaling network.

The epidermal growth factor receptor (EGFR) has been studied extensively because of its critical role in cellular signaling and association with disease. Previous models have elucidated interactions between EGFR and downstream adaptor proteins or showed phenotypes affected by EGFR. However, the link between specific EGFR phosphorylation sites and phenotypic outcomes is still poorly understood. Here, we employed a suite of isogenic cell lines expressing site-specific mutations at each of the EGFR C-terminal phosphorylation sites to interrogate their role in the signaling network and cell biological response to stimulation. Our results demonstrate the resilience of the EGFR network, which was largely similar even in the context of multiple Y-to-F mutations in the EGFR C-terminal tail, while also revealing nodes in the network that have not previously been linked to EGFR signaling. Our data-driven model highlights the signaling network nodes associated with distinct EGF-driven cell responses, including migration, proliferation, and receptor trafficking. Application of this same approach to less-studied RTKs should provide a plethora of novel associations that should lead to an improved understanding of these signaling networks.

Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Activation of E3 ubiquitin ligase WWP2 by non-receptor tyrosine kinase ACK1.

WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is a member of the NEDD4 E3 ubiquitin ligase family. WWP2 ligase activity is regulated by the 2, 3-linker auto-inhibition. Tyrosine phosphorylation of the 2, 3-linker was identified as an activating means for releasing the auto-inhibition of WWP2. However, the tyrosine kinase (TK) for the phosphorylation and activation remains unknown. In this report, we have found that non-receptor TK ACK1 binds to the WW3 domain of WWP2 and phosphorylates WWP2. ACK1 phosphorylates WWP2 at the 2, 3-linker and partially activates the ubiquitination ligase activity. Unexpectedly, tyrosine phosphorylation of the 2, 3-linker seems not a major mode for activation of WWP2, as ACK1 causes much higher activation of the 2, 3-linker tyrosine phosphorylation defective mutants of WWP2 than that of wild-type WWP2. Furthermore, epidermal growth factor (EGF) stimulates tyrosine phosphorylation of WWP2 and this EGF-stimulated phosphorylation of WWP2 is mediated by ACK1. Finally, knockdown of WWP2 by shWWP2 inhibits the EGF-dependent cell proliferation of lung cancer A549 cells, suggesting that WWP2 may function in the EGFR signaling in lung cancer progression. Taken together, our findings have revealed a novel mechanism underlying activation of WWP2.

Aberrant function of pathogenic STAT3 mutant proteins is linked to altered stability of monomers and homodimers.

STAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide-binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

LCK inhibition downregulates YAP activity and is therapeutic in patient-derived models of cholangiocarcinoma.

BACKGROUND & AIMS: There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, Yes-associated protein (YAP), is oncogenic in CCA, but has historically been difficult to target therapeutically. Recently, we described a novel role for the LCK proto-oncogene, Src family tyrosine kinase (LCK) in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity. METHODS: A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft models bearing fibroblast growth factor receptor 2 (FGFR2)-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. RESULTS: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of tested patient-derived organoids demonstrated sensitivity to NTRC 0652-0. CCAs with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In patient-derived xenograft models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth. CONCLUSIONS: A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell lines, and patient-derived organoid and xenograft models. IMPACT AND IMPLICATIONS: Although aberrant YAP activation is frequently seen in CCA, YAP targeted therapies are not yet clinically available. Herein we show that a novel LCK-selective tyrosine kinase inhibitor (NTRC 0652-0) effectively inhibits YAP tyrosine phosphorylation and cotranscriptional activity and is well tolerated and cytotoxic in multiple preclinical models. The data suggest this approach may be effective in CCA with YAP dependence or FGFR2 fusions, and these findings warrant further investigation in phase I clinical trials.

Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH.

The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. Through whole genome sequencing we have uncovered conserved V483M Ptprh mutations in PyMT induced tumors. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while rescue with catalytically dead PTPRH did not. A dose response curve illustrated that two human NSCLC lines with naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Osimertinib treatment of these tumors resulted in a reduction of tumor volume relative to vehicle controls. PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Together these data suggest mutations in PTPRH in NSCLC is inhibitory to PTPRH function, resulting in aberrant EGFR activity and ultimately may result in clinically actionable alterations using existing therapies.

Whole-exome sequencing reveals a comprehensive germline mutation landscape and identifies twelve novel predisposition genes in Chinese prostate cancer patients.

Prostate cancer is the most inheritable cancer with approximately 42% of disease risk attributed to inherited factors by studies of twins, indicating the importance of additional genetic screening to identify predisposition variants. However, only DNA damage repair (DDR) genes have been investigated thoroughly in prostate cancer. To determine the comprehensive germline mutation landscape in Chinese prostate cancer patients, we performed whole exome sequencing in 100 Han Chinese patients with prostate cancer in Hong Kong and identified deleterious germline mutations. A total of 36 deleterious germline variants in 25 genes were identified in 29% patients. Variants were found in eight pathways, including DNA methylation, DDR, and tyrosine-protein kinase. These findings were validated in an independent Chinese cohort of 167 patients with prostate cancer in Shanghai. Seven common deleterious-variant-containing genes were found in discovery cohort (7/25, 28%) and validation cohort (7/28, 25%) with three genes not described before (LDLR, MYH7 and SUGCT) and four genes previously reported (FANCI, ITGA6, PABPC1 and RAD54B). When comparing with that of a cohort of East Asian healthy individuals, 12 non-DDR novel potential predisposition genes (ADGRG1, CHD4, DNMT3A, ERBB3, GRHL1, HMBS, LDLR, MYH7, MYO6, NT5C2, NUP98 and SUGCT) were identified using the discovery and validation cohorts, which have not been previously reported in prostate cancer patients in all ethnic groups. Taken together, this study reveals a comprehensive germline mutation landscape in Chinese prostate cancer patients and discovers 12 novel non-DDR predisposition genes to lay the groundwork for the optimization of genetic screening.

Activity of the nonreceptor tyrosine kinase Ack1 is regulated by tyrosine phosphorylation of its Mig6 homology region.

Ack1 is a proto-oncogenic tyrosine kinase with homology to the tumour suppressor Mig6, an inhibitor of the epidermal growth factor receptor (EGFR). The residues critical for binding of Mig6 to EGFR are conserved within the Mig6 homology region (MHR) of Ack1. We tested whether intramolecular interactions between the Ack1 MHR and kinase domain (KD) are regulated by phosphorylation. We identified two Src phosphorylation sites within the MHR (Y859, Y860). Addition of Src-phosphorylated MHR to the Ack1 KD enhanced enzymatic activity. Co-expression of Src in cells led to increased Ack1 activity; mutation of Y859/Y860 blocked this increase. Collectively, the data suggest that phosphorylation of the Ack1 MHR regulates its kinase activity. Phosphorylation of Y859/Y860 occurs in cancers of the brain, breast, colon, and prostate, where genomic amplification or somatic mutations of Ack1 play a role in disease progression. Our findings suggest that MHR phosphorylation could contribute to Ack1 dysregulation in tumours.

Saturation mutagenesis of a predicted ancestral Syk-family kinase.

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.

Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of O-methyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.