Search

This Technical Note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cell and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates the reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24 min active gradient. In 200 ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45 min run covers ∼90% of the expressed proteome. This complete workflow allows for large swaths of the proteome to be identified and is compatible with diverse sample types.

Telehealth, accessing healthcare and wellness remotely, should be a cost-effective and efficient way for individuals to receive care. The convenience of having a reliable remote collection device for blood tests will facilitate access to precision medicine and healthcare. Herein, we tested a 60-biomarker health surveillance panel (HSP), containing 35 FDA/LDT assays and covering at least 14 pathological states, on 8 healthy individuals' ability to collect their own capillary blood from a lancet finger prick and directly compared it to the traditional phlebotomist venous blood and plasma collection methods. All samples were spiked with 114 stable-isotope-labeled (SIL) HSP peptides and quantitatively analyzed by liquid chromatography-multiple reaction monitoring-mass spectrometry (LC/MRM-MS) scheduled method targeting 466 transitions from 114 HSP peptides and by a discovery data-independent acquisition mass spectrometry (DIA-MS) method. The average peak area ratio (PAR) of the HSP quantifier peptide transitions from all 8 volunteers' capillary blood ( n = 48), venous blood ( n = 48), and matched plasma ( n = 24) was <20% coefficients of variation (CV). Heat map analysis of all 8 volunteers demonstrated that each individual had a unique biosignature. Biological replicates from capillary blood and venous blood clustered within each volunteer in k -means clustering analysis. Pearson statistical analysis of the three biofluids indicated that there was >90% similarity. Discovery DIA-MS analysis of the same samples using a plasma spectral library and a pan-human spectral library identified 1121 and 4661 total proteins, respectively. In addition, at least 122 FDA-approved biomarkers were identified. DIA-MS analysis reproducibly quantitated (<30% CV) ∼600-700 proteins in capillary blood, ∼800 proteins in venous blood, and ∼300-400 proteins in plasma, demonstrating that an expansive biomarker panel is possible with current mass spectrometry technology. Both targeted LC/MRM-MS and discovery DIA-MS analysis of whole blood collected on remote sampling devices are viable options for personal proteome biosignature stratification in precision medicine and precision health.

Competing Interests: Competing interest statement: C.L.S. is employed through Torreyana Corp, San Diego, CA; Blackhawk Genomics, Concord, CA; and Advagenix, Rockville, MD. Volunteer activities of C.L.S. include those for College of American Pathologists, Northfield, IL, and Clinical Laboratory Standards Institute, Wayne, PA. T.J.O. is a Member, Scientific Advisory Committee, MioDx and Integrated Nano-Technologies. A.W.I.L.’s laboratory receives sponsorships from AstraZeneca (HK) Ltd. and MSD (HK) Ltd. for providing selected companion diagnostic tests free to public patients. N.C. is an employee at Quest Diagnostics, Inc. F.A.M. is an employee and stock option holder at Castle Biosciences, Inc. A.B.C. is paid teaching faculty for the American Medical Informatics Association Clinical Informatics Board Review Course and receives small honoraria as well as travel reimbursement to speak at multiple scientific and professional medical society meetings. T.L.P. has been consulted (compensated) for Bio-Rad, Inc. and is Director of Pathology Strategies for the Sturge Weber Foundation (compensated). H.E.W. has employment through Viapath, a majority National Health Service-owned independent pathology service provider; has been a paid faculty member at Kingston University, accepted paid accommodation and subsistence as an invited speaker to Cytocell User Group meeting for the United Kingdom and Ireland, and accepted paid event registration as an invited speaker to Digital Pathology/Global Engage meeting. H.E.W.’s laboratory received scholarship funds from The International Council for Standardization in Haematology for providing JAK2 testing. The other authors (K.F., E.A.B., P.M., N.R.P., K.P.P., B.S., M.S., M.L.G., S.M.M., H.F., and S.O.) do not have any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this article.

Background: Understanding mechanisms underlying specific chemotherapeutic responses in subtypes of cancer may improve identification of treatment strategies most likely to benefit particular patients. For example, triple-negative breast cancer (TNBC) patients have variable response to the chemotherapeutic agent cisplatin. Understanding the basis of treatment response in cancer subtypes will lead to more informed decisions about selection of treatment strategies., Methods: In this study we used an integrative functional genomics approach to investigate the molecular mechanisms underlying known cisplatin-response differences among subtypes of TNBC. To identify changes in gene expression that could explain mechanisms of resistance, we examined 102 evolutionarily conserved cisplatin-associated genes, evaluating their differential expression in the cisplatin-sensitive, basal-like 1 (BL1) and basal-like 2 (BL2) subtypes, and the two cisplatin-resistant, luminal androgen receptor (LAR) and mesenchymal (M) subtypes of TNBC., Results: We found 20 genes that were differentially expressed in at least one subtype. Fifteen of the 20 genes are associated with cell death and are distributed among all TNBC subtypes. The less cisplatin-responsive LAR and M TNBC subtypes show different regulation of 13 genes compared to the more sensitive BL1 and BL2 subtypes. These 13 genes identify a variety of cisplatin-resistance mechanisms including increased transport and detoxification of cisplatin, and mis-regulation of the epithelial to mesenchymal transition., Conclusions: We identified gene signatures in resistant TNBC subtypes indicative of mechanisms of cisplatin. Our results indicate that response to cisplatin in TNBC has a complex foundation based on impact of treatment on distinct cellular pathways. We find that examination of expression data in the context of heterogeneous data such as drug-gene interactions leads to a better understanding of mechanisms at work in cancer therapy response.

Cancer genomic data is continually growing in complexity, necessitating improved methods for data capture and analysis. Tumors often contain multiple therapeutically relevant alterations, and co-occurring alterations may have a different influence on therapeutic response compared to if those alterations were present alone. One clinically important example of this is the existence of a resistance conferring alteration in combination with a therapeutic sensitizing mutation. The JAX Clinical Knowledgebase (JAX-CKB) (https://ckb.jax.org/) has incorporated the concept of the complex molecular profile, which enables association of therapeutic efficacy data with multiple genomic alterations simultaneously. This provides a mechanism for rapid and accurate assessment of complex cancer-related data, potentially aiding in streamlined clinical decision making. Using the JAX-CKB, we demonstrate the utility of associating data with complex profiles comprising ALK fusions with another variant, which have differing impacts on sensitivity to various ALK inhibitors depending on context., Competing Interests: The authors declare no competing interests.

Introduction: Comprehensive cancer genomic profiling provides the opportunity to expose the various molecular aberrations potentially driving tumor progression. Consequently, the identity of these genetic drivers can be utilized to match a patient to the most appropriate targeted therapy, thereby increasing the probability of improved clinical outcome. Despite its capability of informing patient care, the adoption of comprehensive cancer genomic profiling in the clinic has not been widespread. The barriers surrounding its universal acceptance are attributed to both physician and patient perspectives. Areas covered: The following report discusses the various obstacles in place, including those related to clinical utility, education, insurance coverage, and clinical trials, which can deter physicians and patients from utilizing genomic profiling for therapeutic decision-making. Expert commentary: The authors review the recent growth and potential of clinical utility studies over the last two years, provide a suggestive framework for educational support, and comment on the use of social media to enhance clinical trial recruitment.

Introduction: The degree and duration of response to epidermal growth factor receptor (EGFR) inhibitors in EGFR mutated lung cancer are heterogeneous. We hypothesized that the concurrent genomic landscape of these tumors, which is currently unknown in view of the prevailing single gene assay diagnostic paradigm in clinical practice, could play a role in clinical outcomes and/or mechanisms of resistance., Methods: We retrospectively probed our institutional lung cancer database for tumors with EGFR kinase domain mutations that were also evaluated by more comprehensive molecular profiling, and evaluated tumor response to EGFR tyrosine kinase inhibitors (TKIs)., Results: Out of 171 EGFR mutated tumor-patient cases, 20 were sequenced using at least a limited comprehensive genomic profiling platform. 50% harbored concurrent TP53 mutation, 10% PIK3CA mutation, 5% PTEN mutation, among others. The response rate to EGFR TKIs, the median progression-free survival (PFS) to TKIs, the percentage of EGFR-T790M TKI resistance and survival had higher trends in EGFR mutant/TP53 wild-type cases when compared to EGFR mutant/TP53 mutant tumors (all p >0.05 without statistical significance); with a significantly longer median PFS in EGFR-exon 19 deletion mutant/TP53 wild-type cancers treated with 1st generation EGFR TKIs (p=0.035)., Conclusions: Concurrent mutations, specifically TP53, are common in EGFR mutated lung cancer and may alter clinical outcomes. Additional cohorts will be needed to determine if comprehensive molecular profiling adds clinically relevant information to single gene assay identification in oncogene-driven lung cancers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The progression of prostate cancer to castration-resistant prostate cancer (CRPC) is often a result of somatic alterations in the PI3K/Akt/mTOR (mammalian target of rapamycin) pathway, suggesting that therapies targeting this pathway might lead to improved survival and efficacy. Here, we systematically evaluate the results of clinical trials investigating mTOR inhibition in CRPC and utilize preclinical data to predict clinical outcomes., Methods: Trials included in the study were identified through PubMed and via review of conference abstracts cited by relevant review articles. The eligibility of trials was independent of sample size, clinical setting, or date., Results: A total of 14 studies were eligible for qualitative analysis. The clinical setting was variable among studies, and all utilized an allosteric mTOR inhibitor as either a monotherapy or in combination. Molecular criteria were evaluated in three trials. Among most studies, the prostate-specific antigen level declined during treatment, but often increased shortly thereafter. Partial responses to treatment were minimal, and no complete responses were reported. Two studies exploring therapy with an mTOR inhibitor in combination with bicalutamide resulted in minimal efficacy. Overall, allosteric mTOR inhibition was deemed to be inadequate for the treatment of CRPC., Conclusion: Preclinical data suggest that a reciprocal feedback mechanism between PI3K and androgen receptor signaling is a potential mechanism behind the clinical inefficacy of mTOR inhibitors in CRPC, indicating combinatorial targeting of PI3K, mTORC1/2, and the androgen receptor might be more effective. Comprehensive analysis of preclinical data to assess clinical trial targets and efficacy may reduce the number of unproductive trials and identify potentially beneficial combinatorial therapies for resistant disease.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Benign ovarian Brenner tumors often are associated with mucinous cystic neoplasms, which are hypothesized to share a histogenic origin and progression, however, supporting molecular characterization is limited. Our goal was to identify molecular mechanisms linking these tumors. DNA from six Brenner tumors with paired mucinous tumors, two Brenner tumors not associated with a mucinous neoplasm, and two atypical proliferative (borderline) Brenner tumors was extracted from formalin-fixed, paraffin-embedded tumor samples and sequenced using a 358-gene next-generation sequencing assay. Variant calls were compared within tumor groups to assess somatic mutation profiles. There was high concordance of the variants between paired samples (40% to 75%; P < 0.0001). Four of the six tumor pairs showed KRAS hotspot driver mutations specifically in the mucinous tumor. In the two paired samples that lacked KRAS mutations, MYC amplification was detected in both of the mucinous and the Brenner components; MYC amplification also was detected in a third Brenner tumor. Five of the Brenner tumors had no reportable potential driver alterations. The two atypical proliferative (borderline) Brenner tumors both had RAS mutations. The high degree of coordinate variants between paired Brenner and mucinous tumors supports a shared origin or progression. Differences observed in affected genes and pathways, particularly involving RAS and MYC, may point to molecular drivers of a divergent phenotype and progression of these tumors., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Availability of genomic information used in the management of cancer treatment has outpaced both regulatory and reimbursement efforts. Many types of clinical trials are underway to validate the utility of emerging genome-based biomarkers for diagnostic, prognostic, and predictive applications. Clinical trials are a key source of evidence required for US Food and Drug Administration approval of therapies and companion diagnostics and for establishing the acceptance criteria for reimbursement., Content: Determining the eligibility of patients for molecular-based clinical trials and the interpretation of data emerging from clinical trials is significantly hampered by 2 primary factors: the lack of specific reporting standards for biomarkers in clinical trials and the lack of adherence to official gene and variant naming standards. Clinical trial registries need specifics on the mutation required for enrollment as opposed to allowing a generic mutation entry such as, "EGFR mutation." The use of clinical trials data in bioinformatics analysis and reporting is also gated by the lack of robust, state of the art programmatic access support. An initiative is needed to develop community standards for clinical trial descriptions and outcome reporting that are modeled after similar efforts in the genomics research community., Summary: Systematic implementation of reporting standards is needed to insure consistency and specificity of biomarker data, which will in turn enable better comparison and assessment of clinical trial outcomes across multiple studies. Reporting standards will facilitate improved identification of relevant clinical trials, aggregation and comparison of information across independent trials, and programmatic access to clinical trials databases., (© 2015 American Association for Clinical Chemistry.)

Background: Precision medicine in oncology relies on rapid associations between patient-specific variations and targeted therapeutic efficacy. Due to the advancement of genomic analysis, a vast literature characterizing cancer-associated molecular aberrations and relative therapeutic relevance has been published. However, data are not uniformly reported or readily available, and accessing relevant information in a clinically acceptable time-frame is a daunting proposition, hampering connections between patients and appropriate therapeutic options. One important therapeutic avenue for oncology patients is through clinical trials. Accordingly, a global view into the availability of targeted clinical trials would provide insight into strengths and weaknesses and potentially enable research focus. However, data regarding the landscape of clinical trials in oncology is not readily available, and as a result, a comprehensive understanding of clinical trial availability is difficult., Results: To support clinical decision-making, we have developed a data loader and mapper that connects sequence information from oncology patients to data stored in an in-house database, the JAX Clinical Knowledgebase (JAX-CKB), which can be queried readily to access comprehensive data for clinical reporting via customized reporting queries. JAX-CKB functions as a repository to house expertly curated clinically relevant data surrounding our 358-gene panel, the JAX Cancer Treatment Profile (JAX CTP), and supports annotation of functional significance of molecular variants. Through queries of data housed in JAX-CKB, we have analyzed the landscape of clinical trials relevant to our 358-gene targeted sequencing panel to evaluate strengths and weaknesses in current molecular targeting in oncology. Through this analysis, we have identified patient indications, molecular aberrations, and targeted therapy classes that have strong or weak representation in clinical trials., Conclusions: Here, we describe the development and disseminate system methods for associating patient genomic sequence data with clinically relevant information, facilitating interpretation and providing a mechanism for informing therapeutic decision-making. Additionally, through customized queries, we have the capability to rapidly analyze the landscape of targeted therapies in clinical trials, enabling a unique view into current therapeutic availability in oncology.

Entrustable professional activities (EPAs) describe the core work that constitutes a discipline's specific expertise and provide the framework for faculty to perform meaningful assessment of geriatric fellows. This article describes the collaborative process of developing the end-of-training American Geriatrics Society (AGS) and Association of Directors of Geriatric Academic Programs (ADGAP) EPAs for Geriatric Medicine (AGS/ADGAP EPAs). The geriatrics EPAs describes a geriatrician's fundamental expertise and how geriatricians differ from general internists and family practitioners who care for older adults., (© 2014, Copyright the Authors Journal compilation © 2014, The American Geriatrics Society.)

Background: Few studies have examined proxy decision-making regarding end-of-life treatment decisions. Proxy accuracy is defined as whether proxy treatment choices are consistent with the expressed wishes of their index elder. The purpose of this study was to examine proxy accuracy in relation to two family factors that may influence proxy accuracy: perceived family conflict and type of elder-proxy relationship., Methods: Telephone interviews with 202 community-dwelling elders and their proxy decision makers were conducted including the Life-Support Preferences Questionnaire (LSPQ), and a measure of family conflict, and sociodemographic characteristics, including type of relationship., Results: Elder-proxy accuracy was associated with the type of elder-proxy relationship. Adult children demonstrated the lowest elder-proxy accuracy and spousal proxies the highest elder-proxy accuracy. Elder-proxy accuracy was associated with family conflict. Proxies reporting higher family conflict had lower elder-proxy accuracy. No interaction between family conflict and relationship type was revealed., Conclusions: Spousal proxies were more accurate in their substituted judgment than adult children, and proxies who perceive higher degree of family conflict tended to be less accurate than those with lower family conflict. Health care providers should be aware of these family factors when discussing advance care planning.

This article reviews research on end-of-life (EOL) decision-making in general and published guidelines on communicating with patients about EOL treatment options. The literature on EOL decision-making, most of which concerns advance care planning decisions, has identified several factors that influence treatment choices including race, religiosity, current health, and family conflict. This literature also documents widespread lack of understanding about dying and palliative care and fears of abandonment by health care providers. This article reviews guidelines for communicating with patients, stresses the role of prognostication in good decision-making, and provides numerous suggestions for initiating and structuring conversations with patients and families about EOL care.

Background: Because many Americans are decisionally incapacitated at the end of life, the last treatment decisions are often made by family proxies. Family discord concerning end-of-life (EOL) care may not only exacerbate a family crisis but also influence treatment decisions. We tested the extent to which family discord would be associated with preferences for types of end-of-life care, predicting that greater discord would be associated with weaker preferences for palliative care and stronger preferences for life-prolonging care., Setting/subjects: Sixty-eight proxy decision makers for elderly relatives were interviewed in their homes over the telephone., Measurements: The 35-minute telephone interview assessed preferences for four life-prolonging treatments and for palliative care and included a family discord measure, an EOL values scale, and questions about sociodemographic characteristics., Results: Repeated-measures analyses of covariance and regression analyses showed that greater family discord was associated with stronger preferences for life-prolonging treatments and weaker preferences for palliative care, independently of EOL values and sociodemographic characteristics., Conclusions: In counseling patients with life-limiting illnesses and their families, clinicians should explore possible family discord surroundings EOL care. Counseling protocols should be developed and clinicians should be trained in counseling to minimize family conflict.

Ca(v)2.1 and Ca(v)2.2 channels conduct P/Q-type and N-type Ca(2+) currents that initiate neurotransmission and bind SNARE proteins through a synaptic protein interaction (synprint) site. PKC and CaMKII phosphorylate the synprint site and inhibit SNARE protein binding in vitro. Here we identify two separate microdomains that each bind syntaxin 1A and SNAP-25 in vitro and are regulated by PKC phosphorylation at serines 774 and 898 and CaMKII phosphorylation at serines 784 and 896. Activation of PKC resulted in its recruitment to and phosphorylation of Ca(V)2.2 channels, but PKC phosphorylation did not dissociate Ca(V)2.2 channel/syntaxin 1A complexes. Chimeric Ca(V)2.1a channels containing the synprint site of Ca(v)2.2 gain modulation by syntaxin 1A, which is blocked by PKC phosphorylation at the sites identified above. Our results support a bipartite model for the synprint site in which each SNARE-binding microdomain is controlled by a separate PKC and CaMKII phosphorylation site that regulates channel modulation by SNARE proteins.

Objectives: To determine if an educational program can improve knowledge and attitude among ancillary staff on end-of-life care issues in a long-term care facility., Design: A pilot study using a pre- and post-test design prior to and at the completion of an education intervention., Setting: A long-term care facility in suburban Philadelphia that has 150 assisted living beds and 53 nursing home beds., Participants: Long-term care ancillary staff including certified nursing assistants (called "care managers" at this facility), social workers, recreational therapists, and food service workers., Intervention: The intervention was a novel educational program consisting of five in-service lectures with accompanying take home self-study modules for ancillary staff in long-term care entitled "Dignity in Dementia.", Measurements: Ancillary staff attitudes and knowledge on end-of-life issues in dementia were assessed with a knowledge and attitude questionnaire pre- and post-intervention. One-year follow-up questionnaires were administered to assess long-term maintenance of knowledge and attitude changes., Results: Thirty-two ancillary staff completed the pre-intervention questionnaires. Twenty-nine ancillary staff completed the post-intervention questionnaires (90.6%). There was a significant change in the end-of-life knowledge level of the ancillary staff (P =.0270). Specifically, there was a significant change in one question dealing with dementia as a terminal disease (P = .006). There were also significant changes in the average attitude scores of the ancillary staff. (P = .0242). One-year follow-up revealed that both knowledge and attitude changes were maintained., Conclusions: This pilot project demonstrates that a staff educational program on end-of-life care for dementia residents can improve end-of-life knowledge and attitudes among long-term care ancillary staff and that this improvement can be maintained for up to 1 year. This intervention is easily reproducible in the long-term care setting. This project is an important step in helping improve end-of-life care for dementia residents in long-term care settings by improving the knowledge and attitudes of their caregivers.

This chapter addresses key components of screening and preventive care for the older population. The older population is heterogeneous, ranging from the competent, active, well individual to the frail, demented individual. Certain preventive measures are important for all individuals such as counseling on exercise and screening for high blood pressure. However, universal cancer, cholesterol, or dementia screening may not be cost effective and beneficial in all older adults. These preventive measures should be guided by the individuals' circumstances including their life expectancy, co-morbid illnesses, functional capacity, and quality of life. Clinicians may be able to individualize preventive medicine decisions by stratifying their patients into well and frail using the guidelines we have provided. The goal of prevention and screening in older patients is to improve function and quality of life. Primary care physicians should facilitate discussion of preventive measures with their older patients as part of their ongoing health care.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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 (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.

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.

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.

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.