Somatic gene mutation analysis of triple negative breast cancers.

OBJECTIVES: The aims of this study were to analyze triple negative breast cancer (TNBC) using an expanded next generation sequencing (NGS) assay, assess the clinical relevance using a recently described database, and correlate tumor morphology with detected genetic alterations. METHODS: DNA was isolated from twenty primary TNBCs and genes of interest were enriched and sequenced with hybrid capture, followed by variant detection and functional and clinical annotation. The JAX-CTP™ assay detects actionable variants in the form of single nucleotide variations, small insertions and deletions (≤50 bp), and copy number variants in 358 genes in specimens containing a neoplastic cell content of ≥50%. The JAX-CKB is a comprehensive database that curates tumor phenotype, genetic variant and protein effect, therapeutic relevance, and available treatment options. RESULTS: 18/20 (90%) of TNBCs contained at least one somatic mutation detected by the JAX-CTP™. MYC amplification was the most common alteration, present in 75% of tumors. TP53, AURKA, and KDR mutations were each present in 30% (6/20) of cases. Related recruiting clinical trials, extracted from JAX-CKB, included 166 for breast cancer, of which 17 were specific to only the TNBC subtype. All 17 trials were testing at least one therapy that targets a mutation identified in this sample set. The majority (89%) of tumors with basal-like histologic features had MYC amplification. CONCLUSIONS: The expanded gene panel identified a variety of clinically actionable gene alterations in TNBCs. The identification of such variants increases the possibility for new therapeutic interventions and clinical trial eligibility for TNBC patients.

Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase.

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) belongs to the aromatic amino acid hydroxylase superfamily, which includes phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). The crystal structures for both PAH and TH have been reported, but a crystallographic model of TPH remains elusive. For this reason, we have utilized the information presented in the TH crystal structure in combination with primary sequence alignments to design point mutations in potential structural domains of the TPH protein. Mutation of a TH salt bridge (K170E) was sufficient to alter enzyme macromolecular assembly. We found that the disruption of the cognate intersubunit dimerization salt bridge (K111-E223) in TPH, however, did not affect the macromolecular assembly of TPH. Enzyme peaks representing only tetramers were observed with size exclusion chromatography. By contrast, a single-point mutation within the tetramerization domain of TPH (L435A) was sufficient to disrupt the normal homotetrameric assembly of TPH. These studies indicate that, although the proposed salt bridge dimerization interface of TH is conserved in TPH, this hypothetical TPH intersubunit binding domain, K111-E223, is not required for the proper macromolecular assembly of the protein. However, leucine 435 within the tetramerization domain is necessary for the proper macromolecular assembly of TPH.

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21-41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (Ndelta15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion Ndelta15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (Ndelta30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (Ndelta41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.

Advances in the molecular characterization of tryptophan hydroxylase.

The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized into N-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH by L-DOPA may have implications for depression in Parkinson's disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article.

Tyrosine hydroxylase and tryptophan hydroxylase do not form heterotetramers.

Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) both contain a C-terminal tetramerization domain composed of a leucine heptad repeat embedded within a 4,3-hydrophobic repeat. Previous mutagenesis experiments and X-ray crystallographic studies have demonstrated that these repeats are required for tetramer assembly of the hydroxylase enzymes via coiled-coil interactions. The specificity of these particular C-terminal intersubunit binding motifs was investigated by determining if TH and TPH can form heterotetramers when coexpressed in bacteria. Bacterial cells were contransformed with TH and TPH expression plasmids under kanamycin and ampicillin selection, respectively. Immunoprecipitation of induced bacterial supernatants with a TPH monoclonal antibody demonstrated that, unlike the human TH isoforms, TH and TPH do not form heterotetramers. The data suggest that specificity of oligomerization of the aromatic amino acid hydroxylases may be partially determined by polar amino acids interspersed within the coiled-coil. This finding should be influential in the development of eukaryotic expression systems and ultimately in gene therapy approaches.

Carboxyl terminal deletion analysis of tryptophan hydroxylase.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the synthesis of serotonin and participates (in a non-rate-limiting fashion) in melatonin biosynthesis. In rabbit, TPH exists as a tetramer of four identical 51007 dalton (444 amino acids) protein subunits. An intersubunit binding domain responsible for tetramer formation of TPH was identified by assessing the role of a carboxyl terminal leucine heptad and 4-3 hydrophobic repeat. These repeats are conserved in all of the aromatic amino acid hydroxylases and have been shown to be required for the assembly of tyrosine hydroxylase tetramers. Polymerase chain reaction was utilized to create three TPH carboxyl terminal deletions (C delta8, C delta12 and C delta17) that sequentially remove members of the leucine heptad and 4-3 hydrophobic repeat. Each deletion and full-length recombinant TPH was expressed in bacteria to obtain soluble enzyme extracts for subsequent activity and structural analysis. It was found that removal of 8, 12 or 17 amino acids from the carboxyl terminus of TPH did not significantly alter enzymatic activity when compared to full-length recombinant TPH. However, the macromolecular structure of the deletions was dramatically affected as determined by dimeric and monomeric profiles on size exclusion chromatography. It can be concluded that amino acids 428-444 (the C-terminal 17 amino acids) comprise an intersubunit binding domain that is required for tetramer formation of TPH, but that tetramer assembly is not essential for full enzymatic activity.

Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., Ndelta50, Ndelta60, Ndelta90, Ndelta106, and Ndelta116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (Ndelta116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and Ndelta50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.

A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.