Protein Tyrosine Kinase Domain Research Articles

Heterozygous missense CSF1R variants hamper in vitro CD34+-derived dendritic cell generation but not in vivo dendritic cell development

Colony stimulating factor 1 receptor (CSF1R) is an essential receptor for both colony stimulating factor 1 (CSF1) and interleukin (IL) 34 signaling expressed on monocyte precursors and myeloid cells, including monocytes, dendritic cells (DC), and microglia. In humans, dominant heterozygous pathogenic variants in CSF1R cause a neurological condition known as CSF1R-related disorder (CSF1R-RD), typically with late onset, previously referred to as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). CSF1R-RD is characterized by microglia reduction and altered monocyte function; however, the impact of pathogenic CSF1R variants on the human DC lineage remains largely unknown.We previously reported that cord blood CD34+ stem cell-derived DCs generated in vitro originate specifically from CSF1R expressing precursors. In this study, we examined the DC lineage of four unrelated patients with late-onset CSF1R-RD who carried heterozygous missense CSF1R variants (c.2330G>A, c.2375C>A, c.2329C>T, and c.2381T>C) affecting different amino acids in the protein tyrosine kinase domain of CSF1R. CD34+ stem cells and CD14+ monocytes were isolated from peripheral blood and subjected to an in vitro culture protocol to differentiate towards conventional DCs and monocyte-derived DCs, respectively. Flow cytometric analysis revealed that monocytes from patients with late-onset CSF1R-RD were still able to differentiate into monocyte-derived DCs in vitro, whereas the ability of CD34+ stem cells to differentiate into conventional DCs was impaired. Strikingly, the peripheral blood of patients contained all naturally occurring DC subsets. We conclude that the in vitro abrogation of DC-development in patients with heterozygous pathogenic missense CSF1R variants does not translate to an impairment in DC development in vivo and speculate that CSF1R signalling in vivo is compensated, which needs further study.

Conformational Transitions in EGFR Protein Tyrosine Kinase Domain and Their Modulation by Mutants

Conformational Transitions in EGFR Protein Tyrosine Kinase Domain and Their Modulation by Mutants

Medicinal Chemistry Perspectives on Recent Advances in Src Kinase Inhibitors as a Potential Target for the Development of Anticancer Agents: Biological Profile, Selectivity, Structure-Activity Relationship.

The physiological Src proto-oncogene is a protein tyrosine kinase receptor that served as the essential signaling pathway in different types of cancer. Src kinase receptor is divided into different domains: a unique domain, an SH3 domain, an SH2 domain, a protein tyrosine kinase domain, and a regulatory tail, which runs from the N-terminus to the C-terminus. Src kinase inhibitors bind in the kinase domain and are activated by phosphorylation. The etiology of cancer involved various signaling pathways and Src signaling pathways are also involved in those clusters. Although the dysregulation of Src kinase resulted in cancer being discovered in the late 19th century it is still considered a cult pathway because it is not much explored by different medicinal chemists and oncologists. The Src kinase regulated through different kinase pathways (MAPK, PI3K/Akt/mTOR, JAK/STAT3, Hippo kinase, PEAK1, and Rho/ROCK pathways) and proceeded downstream signaling to conduct cell proliferation, angiogenesis, migration, invasion, and metastasis of cancer cells. There are numerous FDA-approved drugs flooded the market but still, there is a huge demand for the creation of novel anticancer drugs. As the existing drugs are accompanied by several adverse effects and drug resistance due to rapid mutation in proteins. In this review, we have elaborated about the structure and activation of Src kinase, as well as the development of Src kinase inhibitors. Our group also provided a comprehensive overview of Src inhibitors throughout the last two decades, including their biological activity, structure-activity relationship, and Src kinase selectivity. The Src binding pocket has been investigated in detail to better comprehend the interaction of Src inhibitors with amino acid residues. We have strengthened the literature with our contribution in terms of molecular docking and ADMET studies of top compounds. We hope that the current analysis will be a useful resource for researchers and provide glimpse of direction toward the design and development of more specific, selective, and potent Src kinase inhibitors.

#3697 FAMILIAL PAPILLARY RENAL CELL CARCINOMA: A CASE REPORT

Abstract Background and Aims The incidence of Renal Cell Carcinoma (RCC) is the 9th commonest cancer worldwide. It originates from the renal tubular epithelium and to date has been subclassified into five different categories. Of which, papillary carcinoma is one subclassification and it includes two variants: sporadic and hereditary. This case report aims to outline a rare case of hereditary papillary RCC and the implications for the family involved. Method We performed a retrospective assessment of clinical and radiological characteristics of the index patient and affected relatives. Genetic diagnosis and management have been summarised. Results A 62-year-old male (II.1) was diagnosed with RCC after presenting with right-sided flank pain and multiple complex cystic lesions within both kidneys. A kidney biopsy revealed a papillary structure with foamy histocytes and clear cell-type epithelium clusters. Immunostaining of tumour cells demonstrated CK7 and P504S positivity consistent with RCC. Subsequently, he underwent bilateral nephrectomies and being rendered anephric, was commenced on haemodialysis. He underwent deceased donor renal transplantation four years later at the age of 66 (HLA 1:2:1, CMV -/-, cRF 35%) and remains dialysis independent six years on. After the diagnosis of the index patient, his son (III.1) was found to have multifocal papillary RCC, having also presented with flank pain, but this time in his 30s (Fig. 1). Patient III.1 underwent genetic testing which revealed a missense variant in the MET gene (NM_000245.4 c.3308G>A; p.Gly1103Glu). The variant affects a highly conserved residue in the Tyrosine Kinase domain of the MET protein. Pathogenic activating variants are associated with RCC. Identification of the same variant in the affected father with bilateral disease allowed classification of the variant as likely pathogenic according to the ACMG criteria [1]. The father has seven children, all of whom have now been tested. 5/7 of them have been found to carry the likely pathogenic MET missense variant. Of the 4 asymptomatic carriers, all are under surveillance by regular renal imaging and (one has had radiofrequency ablation). Conclusion The case outlines a rare form of hereditary RCC and the implications that detection of the genetic variant has meant in terms of early diagnosis of disease. Incidence of RCC is increasing worldwide. Hereditary papillary RCC is rare, it is typically inherited in an autosomal dominant pattern. Germline mutations in the MET proto-oncogene are located at 7q31, which encodes for the tyrosine kinase signaling pathway. Variants lead to uncontrolled activation of MET protein and aberrant cell growth [2]. Discussion Hereditary papillary RCC has an estimated incidence of < 1:1500000, its rarity highlighted by the fact that only ∼35 families have been reported worldwide in literature. In these cases it exhibits 100% penetrance, with patients developing renal cell carcinoma typically between their fifth and sixth decade [3]. We would like to acknowledge the family involved in this case report. As with many cancers, early diagnosis is helped by detection of a genetic variant, leading to improved long-term disease-free survival. In the case of metastatic papillary RCC 2-year survival is still estimated at 18% (3).

Abstract LB006: Interactome analysis identifies signaling pathways activated by ETV6-NTRK3 oncogenic gene fusions

Abstract Chromosomal translocations creating fusion genes are among the most common mutation class of known cancer genes, and they have long been identified as driver mutations in certain types of cancer. Recently, oncogenic fusion genes (oncofusions, OFs) have been found in many hematological and solid tumors, demonstrating that translocations are a common cause of malignancy. The frequency of recurrent gene fusions varies depending of the specific type of cancer, but currently identified translocations are estimated to drive up-to 20% of cancer morbidity. In many occasions the oncofusion is the sole driver of oncogenesis when present and certain oncofusion pairs are typically found in one or few specific cancers. ETV6-NTRK3 (EN) oncofusion, is a product of the chromosomal t(12; 15)(p13; q25) translocation, which fuses the N-terminal SAM (sterile alpha motif) domain of ETV6 to the C-terminal protein tyrosine kinase domain of NTRK3 (also known as TrkC). EN is expressed from the ETV6 promoter in the fused chromosome 15. The ETV6 promoter is generally more active, than NTRK3 promoter, and causes EN fusions to be expressed more highly in several tissues and especially in bone marrow and salivary glands. ETV6 forms fusions with many kinases, which are found in hematological malignancies and solid tumors. Except for ETV6-NTRK3, other ETV6-kinase fusions are reported in either hematological or solid malignancies and ETV6-NTRK3 further distinguishes itself by being also reported in several subtypes in both cases. Four EN variants with alternating break points have since been detected in a wide range of human cancers. To provide insight into EN oncogenesis, we employed a proximity labeling mass spectrometry approach to define the molecular context of the EN fusions. We identify 237 high-confidence interactors, which link EN fusions to several key signaling pathways, including ERBB, Insulin and JAK/STAT. We then assess the effects of EN variants on these pathways, and show that the pan NTRK inhibitor selitrectinib (LOXO-195) inhibits the oncogenic activity of EN2, the most common variant. This systems-level analysis of defines the molecular framework in which EN oncofusions operate to promote cancer. Citation Format: Matias Kinnunen, Xiaonan Liu, Kari Salokas, Salla Keskitalo, Markku Varjosalo. Interactome analysis identifies signaling pathways activated by ETV6-NTRK3 oncogenic gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB006.

PD-L1 expression is regulated by ATP-binding of the ERBB3 pseudokinase domain

How PD-L1 expression is regulated in cancer is poorly understood. Here, we report that the ATP-binding activity of ERBB3 pseudokinase regulates PD-L1 gene expression in colorectal cancers (CRCs). ERBB3 is one of the four members of the EGF receptor family, all with protein tyrosine kinase domains. ERBB3 is a pseudokinase with a high binding affinity to ATP. We showed that ERBB3 ATP-binding inactivation mutant reduces tumorigenicity in genetically engineered mouse models and impairs xenograft tumor growth of CRC cell lines. The ERBB3 ATP-binding mutant cells dramatically reduce IFN-γ-induced PD-L1 expression. Mechanistically, ERBB3 regulates IFN-γ-induced PD-L1 expression through the IRS1-PI3K-PDK1-RSK-CREB signaling axis. CREB is the transcription factor that regulates PD-L1 gene expression in CRC cells. Knockin of a tumor-derived ERBB3 mutation located in the kinase domain sensitizes mouse colon cancers to anti-PD1 antibody therapy, suggesting that ERBB3 mutations could be predictive biomarkers for tumors amenable to immune checkpoint therapy.

Clinical and Molecular Features of FTL3 non Canonical and Activation Loop Mutations

Activating mutations of FLT3 (FLT3MT) gene are one of the most frequent pathogenic lesions in AML. As a consequence, tailored therapeutics have been developed in this setting. For instance, protein tyrosine kinase (PTK) domain is a target of inhibitors, which have already entered clinical practice. Overall, FLT3MT occur in about 30% of AML cases, with the internal tandem duplication (ITD) being by far the most common mutation. Indeed, FLT3ITD account for about 83% of all FLT3MT cases and are associated with dismal prognosis. In addition to FLT3ITD, distinct point mutations and deletions within the tyrosine kinase domain (FLT3TKD) and more specifically its activation loop (FLT3AL) have been described and can be also targeted by PTK inhibitors. Nonetheless, these latter mutations are less common, making up approximately 7-10% of all AML cases. Besides these "canonical” mutation types, point mutations outside of the FLT3AL, or "non-canonical” FLT3MT (FLT3NC) have also been observed in AML and other related myeloid neoplasms (MN). However, the prognostic impact and clinical relevance of such molecular alterations are not fully elucidated. This study was inspired by a case diagnosed with CMML harboring a FLT3NC mutation at the amino acid position 592 substituting an alanine for a valine. This spot is immediately located at the starting of the region (591-597) and is important for maintaining the normal kinase in an inactive state in absence of the ligand. Of note is that treatment with the highly-selective, second generation PTK inhibitor, gilteritinib resulted in a good clinical response. Therefore, we reviewed NGS sequencing results of 5,308 patients with MN in search for cases carrying similar lesions. A total of 248 patients with somatic FLT3MT were identified with 152 FLT3ITD, 66 FLT3AL, and 30 FLT3NC hits (Fig.1). The ITD subgroup included 144 AML, 4 MDS, 2 MPN, and 2 MDS/MPN overlap cases. Within cases with FLT3AL mutations, diagnoses consisted of 61 AML, 2 MDS, and 3 MDS/MPN. Most importantly, we also found 30 cases with FLT3NC mutations including 19 AML, 3 MDS, 1 MPN, 4 MDS/MPN, and notably 3 MDS cases with an antecedent history of aplastic anemia. While all the FLT3AL were predicted to be pathogenic based on the ACMG criteria, the FLT3NC consisted of 7 VUS, 10 likely pathogenic, and 9 known pathogenic variants with one biallelic, two recurrent variants in 2 patients and one (N676K) recurring in 4 patients. These atypical FLT3MT mapping to non-canonical locations were found more commonly in non-AML entities (37% in MDS/MPN, MDS, MPN vs. 63% AML) vs.FLT3AL (92% in AML; P< .001) and FLT3ITD (95% AML; P<.001). Co-mutational burden was evaluated with an NGS panel of 51 commonly mutated genes in MN. As expected, NPM1MT was significantly more likely to be associated with cases carrying FLT3ITD (46%) than FLT3NC (20%) (P= .008). Additionally, DNMT3A was found to be recurrently associated to cases with both FLT3ITD (36%) and FLT3AL (30%) as compared to FLT3NC (10%) (P= .005, P= .023, respectively). Median VAFs of both FLT3ITD and FLT3AL were not different compared to FLT3NC (21 vs. 31%; P= .08). Analysis of clinical phenotypes revealed that FLT3AL was more associated with high-grade diseases compared to FLT3NC (94% vs. 67%; P= .001). However, there was no difference in overall survival (OS) between FLT3AL and FLT3NC in AML (23 vs. 36 months; P= .70). In terms of treatment, 17 patients: 12 FLT3AL and 5 FLT3NC (4 AML, 1 MDS/MPN) received TKI inhibitors. In this limited group, no OS differences between AML patients treated with PTK inhibitors (13 cases receiving midostaurin, 3 gilteritinib and 1 sorafenib) and non-treated patients were observed in either FLT3AL or FLT3NC. However, all 5 patients harboring FTL3NC responded to PTK inhibitors. In sum, the well-studied FLT3ITD and NPM1 association does not extend to point mutations outside the FLT3AL. In addition, although differences were noted in less advanced diseases, once patients progressed to AML no specific features, clinical parameters or prognosis differed between cases with FLT3 point mutations/deletions within the activation loop, which appeared to be similar to patients with other types of point mutations outside this region. We also report on the use of PTKs in FLT3NC, whose therapeutic response in association with such FLT3 molecular patterns should warrant further studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for...

FLT4 Mutations Are Associated with Segmental Lymphatic Dysfunction and Initial Lymphatic Aplasia in Patients with Milroy Disease.

This study explored mutations in the Fms-related tyrosine kinase 4/vascular endothelial growth factor receptor 3 gene (FLT4) and lymphatic defects in patients with Milroy disease (MD). Twenty-nine patients with lower limb lymphedema were enrolled. Sixteen patients had a familial history of MD, while 13 patients exhibited sporadic MD. Clinical signs, FLT4 mutations, indocyanine green (ICG) lymphography findings, and skin tissue immunohistochemical staining results were evaluated. Twenty-eight variants in FLT4 were identified. Twelve of these have previously been reported, while 16 are novel. Of the 28 variants, 26 are missense mutations, and the remaining two comprise a splicing mutation and a non-frame shift mutation. Twenty-five variants are located in the intracellular protein tyrosine kinase domain; three are located in the extracellular immunoglobulin domain. Substantially delayed contrast-enhanced tortuous lymphatic vessels were visualized to the ankle or knee level in 15 of 23 patients who underwent ICG lymphography. No initial lymphatic vessels were visualized in skin specimens from four patients who did not exhibit lymphatic vessels during imaging analyses. No specific variant was identified in relation to the unique clinical phenotype. Segmental dysfunction of lymphatic vessels and initial lymphatic aplasia are present in MD patients with FLT4 mutations.

Mutation profiling of uterine cervical cancer patients treated with definitive radiotherapy

ObjectiveTo elucidate tumor mutation profiles associated with outcomes of uterine cervical cancer (UCC) patients treated with definitive radiotherapy. MethodsNinety-eight patients with newly diagnosed and pathologically confirmed UCC (82 squamous cell carcinomas, 12 adenocarcinomas, and four adenosquamous carcinomas) who were treated with definitive radiotherapy were analyzed. DNA was extracted from pre-treatment tumor biopsy specimens. The exons of 409 cancer-related genes were sequenced using a next-generation sequencer. Genetic mutations were identified and analyzed for correlations with clinical outcome. ResultsRecurrent mutations were observed in PIK3CA (35.7%), ARID1A (25.5%), NOTCH1 (19.4%), FGFR3 (16.3%), FBXW7 (19.4%), TP53 (13.3%), EP300 (12.2%), and FGFR4 (10.2%). The prevalence of mutations in FGFR family genes (i.e., FGFR1–4) was almost as high (24.5%) as that in PIK3CA and ARID1A, both of which are well-studied drivers of UCC. Fifty-five percent (21 of 38) of the identified FGFR mutations were located in the FGFR protein tyrosine kinase domain. Five-year progression-free survival (PFS) rates for FGFR mutation-positive patients (n = 24) were significantly worse than those for FGFR mutation-negative patients (n = 74) (43.9% vs. 68.5%, respectively; P = 0.010). Multivariate analysis identified FGFR mutations as significant predictors of worse 5 year PFS (P = 0.005), independent of clinicopathological variables. ConclusionsFGFR mutations are associated with worse PFS in UCC patients treated with definitive radiotherapy. These results warrant further validation in prospective studies.

Endoplasmic reticulum stress causes insulin resistance by inhibiting delivery of newly synthesized insulin receptors to the cell surface.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signaling network known as the unfolded protein response (UPR). Here we characterize how ER stress and the UPR inhibit insulin signaling. We find that ER stress inhibits insulin signaling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesized insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signaling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible FV2E dimerization domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signaling events in the UPR, such as activation of the JNK mitogen-activated protein (MAP) kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signaling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesized insulin receptors in ER-stressed cells.

Discovery of Berberine that Targetedly Induces Autophagic Degradation of both BCR-ABL and BCR-ABL T315I through Recruiting LRSAM1 for Overcoming Imatinib Resistance.

Imatinib, the breakpoint cluster region protein (BCR)/Abelson murine leukemia viral oncogene homolog (ABL) inhibitor, is widely used to treat chronic myeloid leukemia (CML). However, imatinib resistance develops in many patients. Therefore, new drugs with improved therapeutic effects are urgently needed. Berberine (BBR) is a potent BCR-ABL inhibitor for imatinib-sensitive and -resistant CML. Protein structure analysis and virtual screening were used to identify BBR targets in CML. Molecular docking analysis, surface plasmon resonance imaging, nuclear magnetic resonance assays, and thermoshift assays were performed to confirm the BBR target. The change in BCR-ABL protein expression after BBR treatment was assessed by Western blotting. The effects of BBR were assessed in vitro in cell lines, in vivo in mice, and in human CML bone marrow cells as a potential strategy to overcome imatinib resistance. We discovered that BBR bound to the protein tyrosine kinase domain of BCR-ABL. BBR inhibited the activity of BCR-ABL and BCR-ABL with the T315I mutation, and it also degraded these proteins via the autophagic lysosome pathway by recruiting E3 ubiquitin-protein ligase LRSAM1. BBR inhibited the cell viability and colony formation of CML cells and prolonged survival in CML mouse models with imatinib sensitivity and resistance. The results show that BBR directly binds to and degrades BCR-ABL and BCR-ABL T315I via the autophagic lysosome pathway by recruiting LRSAM1. The use of BBR is a new strategy to improve the treatment of patients with CML with imatinib sensitivity or resistance.See related commentary by Elf, p. 3899.

Genomic profiling of microRNA target genes in colorectal cancer.

Colorectal cancer is the second and third most common cancer in men and women, respectively, worldwide. Alterations such as genetic and epigenetic are common in colorectal cancer and are the basis of tumor formation. The exploration of the molecular basis of colorectal cancer can drive a better understanding of the disease as well as guide the prognosis, therapeutics, and disease management. This study is aimed at investigating the genetic mutation profile of five candidate microRNAs (hsa-miR-513b-3p, hsa-miR-500b-3p, hsa-miR-500a-3p, hsa-miR-450b-3p, hsa-miR-193a-5p) targeted by seven genes (APC, KRAS, TCF7L2, EGFR, IGF1R, CASP8, and GNAS)) using in silico approaches. Two datasets (dataset 1 from our previous study and dataset two (The Cancer Genome Atlas, Nature 2012) were considered for this study. Protein-protein interaction, expression analysis, and genetic profiling were carried out using STRING, FireBrowse, and cBioPortal, respectively. Protein-protein interaction network showed that epidermal growth factor receptor has the highest connection among the target genes and this can be considered as the hub gene. Relative to other solid tumors, in colorectal cancer, six of the target genes were downregulated and only CASP8 was upregulated. Genes with protein tyrosine kinases domain were frequently altered in colorectal cancer and the most common alteration in these genes/domain are missense mutation. These results could serve as a lead in the identification of driver genes responsible for colorectal cancer initiation and progression. However, the intense mechanism of these results remains unclear and further experimental validation and molecular approaches are the focal points in the nearest future.

Loratadine, an antihistamine drug, exhibits anti-inflammatory activity through suppression of the NF-kB pathway

Loratadine is an antihistamine drug that shows promise as an anti-inflammatory drug, but supportive studies are lacking. We elucidated the effects and mechanisms by which loratadine inhibits inflammatory responses. Molecular components were evaluated in macrophages by nitric oxide assay, polymerase chain reaction, luciferase assay, immunoblotting, overexpression strategies and cellular thermal shift assay. At the molecular level, loratadine reduced the levels of nitric oxide, iNOS, IL-1β, TNF-α, IL-6, and COX-2 in RAW264.7 cells treated with lipopolysaccharide. Loratadine also specifically inhibited the NF-kB pathway, targeting the Syk and Src proteins. Furthermore, loratadine bound Src in the bridge between SH2 and SH3, and bound Syk in the protein tyrosine kinase domain. The NF-kB signaling pathway was assessed along with putative binding sites through a docking approach. The anti-inflammatory effect of loratadine was tested using mouse models of gastritis, hepatitis, colitis, and peritonitis. Stomach tissue histopathology, liver morphology, and colon length in the loratadine group were improved over the group without loratadine treatment. Taken together, loratadine inhibited the inflammatory response through the NF-kB pathway by binding with the Syk and Src proteins.

Genome-wide identification and expression profiling of genes encoding universal stress proteins (USP) identify multi-stress responsive USP genes in Arabidopsis thaliana

Universal stress proteins (USPs) are stress-responsive proteins conserved among various organisms, including bacteria, plants and metazoans. However, in plants, the function of most of the USPs remains largely unknown. In the present study, we have identified 53 USP domain-containing proteins encoded by 41 genes in the Arabidopsis genome. Based on the presence of additional protein kinase or tyrosine kinase domain, the nomenclature has been provided to these proteins. Comprehensive in silico expression profiling of AtUSPs under various developmental stages revealed that most of the genes are expressed in a tissue-specific manner. Under abiotic stresses, AtUSP9 and AtUSP12 were identified as multi-stress responsive in both shoot and root tissues. Interestingly, AtUSP9 was also induced under various pathogens and elicitor treatments. The expression analysis of USP genes under abiotic stresses using qRT-PCR correlated well with in silico expression analysis. Thus, the present study provides a blueprint for the functional characterization of AtUSPs to ascertain their role under stress conditions. Moreover, AtUSP9 and AtUSP12 may also be used to engineer plants with improved tolerance against multiple stresses.

High-resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase.

Fes and Fes-related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N-terminal Fer/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, a central Src homology 2 (SH2) domain, and a C-terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine-containing ligands to the SH2 domain. Here, the X-ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D-E-pY-E-N-V-D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer-phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β-turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.

Establishing a molecular protocol for detection of EGFR mutations in patients with non-small cell lung cancer

Lung cancer is one of the quickest and most fatal diseases and is divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Approximately 50% of NSCLC patients harbour mutations in the epidermal growth factor receptor(EGFR) gene, spanning from exon 18 to exon 21, which are responsible for the tyrosine kinase domain of the EGFR protein. Currently, gefitinib and erlotinib are two drugs inhibiting EGFR which are used in the treatment of lung cancer. The indication for use of these drugs depends on the mutation status of EGFR. Resistance to treatment with these two drugs has been found with mutations in exon 20; however, mutations in exon 18, 19, 21 benefited from the same treatment. Therefore, we built a molecular protocol for detecting mutations in exon 21 of EGFR in NSCLC patients in Vietnam which, in this study, is based on the real-time polymerase chain reaction high resolution melting (PCR HRM) technique. We successfully designed pairs of primers for the real-time PCR HRM technique to detect mutations in exon 21 and for the Sanger sequencing method to confirm the real-time PCR HRM results. We also investigated the optimal concentration of MgCl2 for the real-time PCR HRM protocol. Performance characteristics of the real-time PCR HRM protocol were evaluated in terms of specificity, limit of detection, repeatability, and it showed good results. Finally, we applied the molecular protocol to 172 clinical samples and compared these with the Sanger nucleotide sequencing method and the peptide nucleic acid (PNA) clamping method. The results of the comparison demonstrate that the molecular protocol constructed for detection of exon 21 mutations can be used in the treatment of NSCLC patients.

EPHA2 sequence variants are associated with susceptibility to Kaposi’s sarcoma-associated herpesvirus infection and Kaposi’s sarcoma prevalence in HIV-infected patients

BackgroundTo determine if variations exist in the KSHV host receptor EPHA2′s coding region that affect KSHV infectivity and/or KS prevalence among South African HIV-infected patients. MethodsA retrospective candidate gene association study was performed on 150 patients which were randomly selected from a total of 756 HIV-infected patients and grouped according to their KS status and KSHV serodiagnosis; namely group 1: KS+/KSHV+; group 2: KS−/KSHV+; group 3: KS−/KSHV−. Peripheral blood DNA was used to extract DNA and PCR amplify and sequence the entire EPHA2 coding region, which was compared to the NCBI reference through multiple alignment. Results100% (95% CI 92.9–100%) of the KS positive patients, and 31.6% (95% CI 28.3–35.1%) of the KS negative patients were found to be KSHV seropositive. Aggregate variation across the entire EPHA2 coding region identified an association with KS (OR = 6.6 (95% CI 2.8, 15.9), p = 2.2 × 10−5). This was primarily driven by variation in the functionally important protein tyrosine kinase domain (Pkinase-Tyr; OR = 4.9 (95% CI 1.9, 12.4), p = 0.001) and the sterile-α-motif (SAM; OR = 13.8 (95% CI 1.7, 111.6), p = 0.014). Mutation analysis revealed two novel, non-synonymous heterozygous variants (c.2254 T > C: OR undefined, adj. p = 0.02; and c.2990 G > T: OR undefined, adj. p = 0.04) in Pkinase-Tyr and SAM, respectively, to be statistically associated with KS; and a novel heterozygous transition (c.2727C > T: OR = 6.4 (95% CI 1.4, 28.4), adj. p = 0.03) in Pkinase-Tyr to be statistically associated with KSHV. ConclusionsVariations in the KSHV entry receptor gene EPHA2 affected susceptibility to KSHV infection and KS development in a South African HIV-infected patient cohort.

Human epidermal receptor family inhibitors in patients with ERBB3 mutated cancers: Entering the back door

IntroductionTherapeutic inhibition of the human epidermal receptor 3 (ERBB3, HER3) has been challenged by the low frequency of ERBB3 somatic alterations across cancer types. We have evaluated the clinical utility to use available inhibitors of the HER family in the context of ERBB3 mutations. Patients and methodsIn this study, we have selected patients with somatic ERBB3 alterations detected in their tumours from the molecular screening programs running at our institution. Techniques used for molecular screening were targeted next generation sequencing, comparative genomic hybridisation and/or whole exome sequencing on fresh frozen tumour biopsies. ResultsOn the 844 patients with a molecular portrait of their tumours, 31 (3.7%) had a somatic mutation of ERBB3. Overall, 9 patients received available inhibitors of HER family, including trastuzumab and/or lapatinib or afatinib. Sixteen patients received other molecularly targeted agents, including the Mammalian Target Of Rapamycin (mTOR), the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase (PI3K) or NOTCH inhibitors or chemotherapy, and 4 patients failed being treated. Thirteen different histological subtypes were affected by ERBB3 mutations; top ones were colorectal carcinomas (6 patients), non-small cell lung cancers (4 patients, including a squamous cell carcinoma), head and neck squamous cell carcinomas (3 patients) and breast carcinomas (3 patients). The presence of a mutation in the tyrosine kinase domain of the ERBB3/HER3 protein detected in four patients' tumours was significantly related to good progression-free survival (hazard ratio [HR] = 0.18, p value = 0.022) and overall survival (HR = 0.19, p value = 0.03) in univariate analysis. Treatment of these four patients included at least a tyrosine kinase inhibitor lapatinib or afatinib. ConclusionOur exploratory analysis suggests that mutations in the tyrosine kinase domain of the HER3 protein are related to a favourable outcome under HER family inhibitors.

Structure and Dynamics of Membrane Proteins and Membrane Associated Proteins with Native Bicelles from Eukaryotic Tissues.

In vitro studies of protein structure, function, and dynamics typically preclude the complex range of molecular interactions found in living tissues. In vivo studies elucidate these complex relationships, yet they are typically incompatible with the extensive and controlled biophysical experiments available in vitro. We present an alternative approach by extracting membranes from eukaryotic tissues to produce native bicelles to capture the rich and complex molecular environment of in vivo studies while retaining the advantages of in vitro experiments. Native bicelles derived from chicken egg or mouse cerebrum tissues contain a rich composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysolipids, cholesterol, ceramides (CM), and sphingomyelin (SM). The bicelles also contain source-specific lipids such as triacylglycerides (TAGs) and sulfatides from egg and brain tissues, respectively. With the influenza hemagglutinin fusion peptide (HAfp) and the C-terminal Src homology domain of lymphocyte-specific protein-tyrosine kinase (lck-cSH2), we show that membrane proteins and membrane associated proteins reconstituted in native bicelles produce high-resolution NMR data and probe native protein-lipid interactions.