Mig6 not only inhibits EGFR and HER2 but also targets HER3 and HER4 in a differential specificity: Implications for targeted esophageal cancer therapy.

The human EGF receptor family plays pivotal roles in physiology and cancer, which contains four closely-related members: HER1/EGFR, HER2, HER3 and HER4. Previously, it was found that the mitogen-inducible gene 6 (Mig6) protein is a negative regulator of EGFR and HER2 by using its S1 segment to bind at the kinase dimerization interface. However, it is still unclear whether the S1 segment can also effectively target HER3 and HER4? Here, we performed a systematic investigation to address this issue. The segment can bind to all the four HER kinases with a varying affinity and moderate selectivity; breaking of the segment into shorter hotspot peptides would largely impair the affinity and selectivity, indicating that the full-length sequence is required for the effective binding of S1 to these kinases. The hs2 peptide, which corresponds to the middle hotspot region of S1 segment, can partially retain the affinity to HER kinases, can moderately compete with S1 segment at the dimerization interfaces, and can mimic the biological function of Mig6 protein to suppress HER4+ esophageal cancer at cellular level. In addition, we also analyzed the binding potency of S1 segment and hs2 peptide to the kinase domains of other five widely documented growth factor receptors (GFRs). It was showed that both the S1 and hs2 cannot effectively interact with these receptors. Overall, the Mig6 is suggested as a specific pan-HER inhibitor, which can target and suppress HER family members with a broad selectivity, but exhibits weak or no activity towards other GFRs.

The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease.

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism.

ErbB4 Mutation that Decreased NRG1-ErbB4 Signaling Involved in the Pathogenesis of Amyotrophic Lateral Sclerosis/Frontotemporal Dementia.

BACKGROUND: Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) includes a large spectrum of neurodegenerative disorders. OBJECTIVE: To identify the relationship of ErbB4 mutation and ALS/FTD. METHODS: Here, we report an atypical case of frontal variant behavioral abnormalities at the initial stage, a stable plateau stage of 5 years, and paralysis involving both upper and lower motor neurons followed by progressive cognitive dysfunction at the advanced stage. The clinical findings suggested a diagnosis of ALS/FTD, and genetic testing revealed erb-b2 receptor tyrosine kinase 4 (ErbB4) heterozygous mutation (c.2136 T>G, p.I712M), identified in an ALS pedigree previously. We modeled mutant ErbB4 protein through the SWISS-MODEL Server, and speculated on the structural change caused by the mutation. We also identified that ErbB4 (I712M) mutation led to reduced auto-phosphorylation of ErbB4 upon neuregulin-1 (NRG1) stimulation. RESULTS: A functional analysis of ErbB4 mutation demonstrated an obviously decreased auto-phosphorylation of ErbB4 involving in the pathogenesis of ALS/FTD. CONCLUSION: We firstly found ErbB4 mutation to be identified in ALS/FTD.

Negative feedback regulation of ErbB4 tyrosine kinase activity by ERK-mediated non-canonical phosphorylation.

ErbB4 receptor tyrosine kinase has four different isoforms that are classified based on variants in the extracellular juxtamembrane domain (JM-a and JM-b) and the C-terminal region (CYT-1 and CYT-2). Here, we used the JM-b/CYT-1 isoform to investigate the roles of serine/threonine phosphorylation in MEK-ERK-dependent feedback inhibition. TPA as an activator of the ERK pathway markedly induced ErbB4 phosphorylation at Thr-674, the conserved common feedback site in the intracellular JM domain, which resulted in the downregulation of tyrosine autophosphorylation. We also identified Ser-1026 as an ErbB4-specific ERK target site in the CYT-1 region. Moreover, double mutations (Thr-674/Ser-1026 to Ala) significantly upregulated ErbB4 activation, indicating that Thr-674 and Ser-1026 are cooperatively involved in negative feedback regulation. Given the fact that ErbB4 mutation is one of the most common genetic alterations in melanoma cells, we demonstrated that a typical oncogenic ErbB4 mutant was resistant to the negative feedback regulation to maintain a highly active status of tyrosine kinase activity. Together, these findings indicate that feedback mechanisms are key switches determining oncogenic potentials of ErbB receptor kinases.

Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection.

A novel Affimer-functionalised interdigitated electrode-based capacitive biosensor platform was developed for detection and estimation of Her4, a protein tumour biomarker, in undiluted serum. An anti-Her4 Affimer with a C-terminal cysteine was used to create the bio-recognition layer via self-assembly on gold interdigitated electrodes for the sensor fabrication. Electrochemical impedance spectroscopy (EIS) in the absence of redox markers was used to evaluate the sensor performance by monitoring the changes in capacitance. The Affimer sensor in buffer and in undiluted serum demonstrated high sensitivity with a broad dynamic range from 1 pM to 100 nM and a limit of detection lower than 1 pM both in buffer and in serum. Furthermore, the Affimer sensor demonstrated excellent specificity with negligible interference from serum proteins, suggesting resilience to non-specific binding. The sensing ability of the present Affimer sensor in spiked undiluted serum suggests its potential for a new range of Affimer-based sensors. The fabricated Affimer sensor can thus be further adapted with other probes having affinities to other biomarkers for a new range of biosensors.

Identification and characterization of ErbB4 kinase inhibitors for effective breast cancer therapy.

The overexpression of ErbB4 is associated with aggressive disease biology and reduced the survival of breast cancer patients. We have used ErbB4 receptor as a novel drug target to spearhead the rational drug design. The present study is divided into two parts. In the first part, we have exploited the hidden information inside ErbB4 kinase receptor both at sequence and structural level. PSI-BLAST algorithm is used to search similar sequences against ErbB4 kinase sequence. Top 15 sequences with high identity were selected for finding conserved and variable regions among sequences using multiple sequence alignment. In the second part, available 3 D structure of ErbB4 kinase is curated using loop modeling, and anomalies in the modeled structure is improved by energy minimization. The resultant structure is validated by analyzing dihedral angles by Ramachandran plot analysis. Furthermore, the potential binding sites were detected by using DoGSite and CASTp server. The similarity-search criterion is used for the preparation of our in-house database of drugs from DrugBank database. In total, 409 drugs yet to be tested against ErbB4 kinase is used for screening purpose. Virtual screening results in identification of 11 compounds with better binding affinity than lapatinib and canertinib. Study of protein-ligand interactions reveals information about amino acid residues; Lys726, Thr771, Met774, Cys778, Arg822, Thr835, Asp836 and Phe837 at the binding pocket. The physicochemical properties and bioactivity score calculation of selected compounds suggest them as biological active. This study presents a rich array that assist in expediting new drug discovery for breast cancer.

ErbB1 and ErbB4 generate opposing signals regulating mesenchymal cell proliferation during valvulogenesis.

Heparin-binding EGF-like growth factor (HB-EGF) plays an indispensable role in suppression of cell proliferation during mouse valvulogenesis. However, ligands of the EGF receptor (EGFR/ErbB1), including HB-EGF, are generally considered as growth-promoting factors, as shown in cancers. HB-EGF binds to and activates ErbB1 and ErbB4. We investigated the role of ErbB receptors in valvulogenesis in vivo using ErbB1- and ErbB4-deficient mice, and an ex vivo model of endocardial cushion explants. We show that HB-EGF suppresses valve mesenchymal cell proliferation through a heterodimer of ErbB1 and ErbB4, and an ErbB1 ligand (or ligands) promotes cell proliferation through a homodimer of ErbB1. Moreover, a rescue experiment with cleavable or uncleavable isoforms of ErbB4 in ERBB4-null cells indicates that the cleavable JM-A, but not the uncleavable JM-B, splice variant of ErbB4 rescues the defect of the null cells. These data suggest that the cytoplasmic intracellular domain of ErbB4, rather than the membrane-anchored tyrosine kinase, achieves this suppression. Our study demonstrates that opposing signals generated by different ErbB dimer combinations function in the same cardiac cushion mesenchymal cells for proper cardiac valve formation.

Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the ß-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression.

Analysis of regulatory mechanism after ErbB4 gene mutation based on local modeling methodology.

ErbB4 is an oncogene belonging to the epidermal growth factor receptor family and contributes to the occurrence and development of multiple cancers, such as gastric, breast, and colorectal cancers. Therefore, studies of the regulation of ErbB4 in cancerigenic pathway will advance molecular targeted therapy. Advanced bioinformatic analysis softwares, such as ExPASy, Predictprotei, QUARK, and I-TASSER, were used to analyze the regulatory mechanism after ErbB4 gene mutation in terms of amino acid sequence, primary, secondary, and tertiary structure of the protein and upstream-downstream receptor/ligands. Mutation of the 19th and 113th amino acids at the carboxyl terminus of ErbB4 protein did not affect its biological nature, but its secondary structure changed and protein binding sites were near 2 mutational sites; moreover, after mutation introduction, additional binding sites were observed. Tertiary structure modeling indicated that local structure of ErbB4 was changed from an α helical conformation into a ß chain folding structure; the α helical conformation is the functional site of protein, while active sites are typically near junctions between helical regions, thus the helical structures are easily destroyed and change into folding structures or other structures after stretching. Mutable sites of ErbB4 is exact binding sites where dimer formed with other epidermal growth factor family proteins; mutation enabled the ErbB4 receptor to bind to neuregulin 1 ligand without dimer formation, disrupting the signal transduction pathway and affecting ErbB4 function.

Activating ERBB4 mutations in non-small cell lung cancer.

Recent efforts to comprehensively characterize the mutational landscape of non-small cell lung cancer have identified frequent mutations in the receptor tyrosine kinase ERBB4. However, the significance of mutated ERBB4 in non-small cell lung cancer remains elusive. Here, we have functionally characterized nine ERBB4 mutations previously identified in lung adenocarcinoma. Four out of the nine mutations, Y285C, D595V, D931Y and K935I, were found to be activating, increasing both basal and ligand-induced ErbB4 phosphorylation. According to structural analysis, the four activating mutations were located at critical positions at the dimerization interfaces of the ErbB4 extracellular (Y285C and D595V) and kinase (D931Y and K935I) domains. Consistently, the mutations enhanced ErbB4 dimerization and increased the trans activation in ErbB4 homodimers and ErbB4-ErbB2 heterodimers. The expression of the activating ERBB4 mutants promoted survival of NIH 3T3 cells in the absence of serum. Interestingly, serum starvation of NIH 3T3 cells expressing the ERBB4 mutants only moderately increased the phosphorylation of canonical ErbB signaling pathway effectors Erk1/2 and Akt as compared with wild-type ERBB4. In contrast, the mutations clearly enhanced the proteolytic release of signaling-competent ErbB4 intracellular domain. These results suggest the presence of activating driver mutations of ERBB4 in non-small cell lung cancer.

ERBB4 promoter polymorphism is associated with poor distant disease-free survival in high-risk early breast cancer.

A number of genetic variants have been linked to increased risk of breast cancer. Little is, however, known about the prognostic significance of hereditary factors. Here, we investigated the frequency and prognostic significance of two ERBB4 promoter region variants, -782G>T (rs62626348) and -815A>T (rs62626347), in a cohort of 1010 breast cancer patients. The frequency of nine previously described somatic ERBB4 kinase domain mutations was also analyzed. Clinical material used in the study consisted of samples from the phase III, adjuvant, FinHer breast cancer trial involving 1010 women. Tumor DNA samples were genotyped for ERBB4 variants and somatic mutations using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Paraffin-embedded tumor sections from all patients were immunohistochemically stained for ErbB4 expression. Association of ERBB4 genotype to distant disease-free survival (DDFS) was assessed using Kaplan-Meier and Cox regression analyses. Genotyping was successful for 91-93% of the 1010 samples. Frequencies observed for the ERBB4 variants were 2.5% and 1.3% for -782G>T and -815A>T, respectively. Variant -815A>T was significantly associated with poor survival (HR  = 2.86 [95% CI 1.15-6.67], P = 0.017). In contrast, variant -782G>T was associated with well-differentiated cancer (P = 0.019). Two (0.2%) ERBB4 kinase domain mutations were found, both of which have previously been shown to be functional and promote cancer cell growth in vitro. These data present the germ-line ERBB4 variant -815A>T as a novel prognostic marker in high-risk early breast cancer and indicate the presence of rare but potentially oncogenic somatic ERBB4 mutations in breast cancer.

Molecular basis of the binding of YAP transcriptional regulator to the ErbB4 receptor tyrosine kinase.

The newly discovered transactivation function of ErbB4 receptor tyrosine kinase is believed to be mediated by virtue of the ability of its proteolytically-cleaved intracellular domain (ICD) to physically associate with YAP2 transcriptional regulator. In an effort to unearth the molecular basis of YAP2-ErbB4 interaction, we have conducted a detailed biophysical analysis of the binding of WW domains of YAP2 to PPXY motifs located within the ICD of ErbB4. Our data show that the WW1 domain of YAP2 binds to PPXY motifs within the ICD in a differential manner and that this behavior is by and large replicated by the WW2 domain. Remarkably, while both WW domains absolutely require the integrity of the PPXY consensus sequence, non-consensus residues within and flanking this motif do not appear to be critical for binding. In spite of this shared mode of binding, the WW domains of YAP2 display distinct conformational dynamics in complex with PPXY motifs derived from ErbB4. Collectively, our study lends new insights into the molecular basis of a key protein-protein interaction involved in a diverse array of cellular processes.