Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.

BACKGROUND: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. METHODS: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. RESULTS: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. CONCLUSIONS: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs.

Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants.

BACKGROUND: Pathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well. METHODS: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell-derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament-directed calcium reporter to monitor changes in myofilament calcium affinity. RESULTS: Hypertrophic cardiomyopathy-associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction. TNNT2 variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, HOPX, and NPPB. We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. On the basis of a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 variants of uncertain significance. CONCLUSIONS: Our study found that hypertrophic cardiomyopathy-associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity.

Autotitrating external positive end-expiratory airway pressure to abolish expiratory flow limitation during tidal breathing in patients with severe COPD: a physiological study.

BACKGROUND: The optimal noninvasive application of external positive end-expiratory pressure (EPAP) to abolish tidal-breathing expiratory flow limitation (EFLT) and minimise intrinsic positive end-expiratory pressure (PEEPi) is challenging in COPD patients. We investigated whether auto-titrating EPAP, using the forced oscillation technique (FOT) to detect and abolish EFLT, would minimise PEEPi, work of breathing and neural respiratory drive (NRD) in patients with severe COPD. METHODS: Patients with COPD with chronic respiratory failure underwent auto-titration of EPAP using a FOT-based algorithm that detected EFLT. Once optimal EPAP was identified, manual titration was performed to assess NRD (using diaphragm and parasternal intercostal muscle electromyography, EMGdi and EMGpara, respectively), transdiaphragmatic inspiratory pressure swings (ΔP di), transdiaphragmatic pressure-time product (PTPdi) and PEEPi, between EPAP levels 2 cmH2O below to 3 cmH2O above optimal EPAP. RESULTS: Of 10 patients enrolled (age 65±6 years; male 60%; body mass index 27.6±7.2 kg.m-2; forced expiratory volume in 1 s 28.4±8.3% predicted), eight had EFLT, and optimal EPAP was 9 (range 4-13) cmH2O. NRD was reduced from baseline EPAP at 1 cmH2O below optimal EPAP on EMGdi and at optimal EPAP on EMGpara. In addition, at optimal EPAP, PEEPi (0.80±1.27 cmH2O versus 1.95± 1.70 cmH2O; p<0.05) was reduced compared with baseline. PTPdi (10.3±7.8 cmH2O·s-1 versus 16.8±8.8 cmH2O·s-1; p<0.05) and ΔP di (12.4±7.8 cmH2O versus 18.2±5.1 cmH2O; p<0.05) were reduced at optimal EPAP+1 cmH2O compared with baseline. CONCLUSION: Autotitration of EPAP, using a FOT-based algorithm to abolish EFLT, minimises transdiaphragmatic pressure swings and NRD in patients with COPD and chronic respiratory failure.

Phenotype of Parathyroid-targeted Cdc73 Deletion in Mice Is Strain-dependent.

Hyperparathyroidism jaw-tumor syndrome is an autosomal dominant disorder caused by mutations in the CDC73/HRPT2 tumor suppressor gene, encoding parafibromin, and manifesting benign or malignant parathyroid tumors, ossifying jaw fibromas, uterine tumors, and kidney lesions. Sporadic parathyroid carcinomas also frequently exhibit inactivating CDC73 mutations and loss of parafibromin. To study the role of CDC73 in parathyroid cell proliferation in vivo, we generated mice with a parathyroid-specific deletion of Cdc73. Homozygous knockout mice on a mixed B6/129/CD1 background had decreased serum calcium and PTH and smaller parathyroid glands compared with heterozygous or wild-type littermates, whereas homozygous Cdc73-null mice on other backgrounds exhibited no abnormalities in parathyroid gland function or development. No hypercalcemia or parathyroid hypercellularity was observed in mice of any background examined at any age. Thus, although postnatally acquired complete loss of CDC73 causes parathyroid cell proliferation and hyperparathyroidism, such as seen in human hyperparathyroidism jaw-tumor syndrome, our results suggest that earlier, developmentally imposed complete loss of Cdc73 can cause a primary defect in parathyroid gland structure/function in a strain-dependent manner. This striking disparity in parathyroid phenotype related to genetic background offers a unique opportunity in an in vivo model system to precisely dissect and identify the responsible molecular mechanisms.

Improving Access and Quality of Behavioral Health Services for Health Care Employees.

OBJECTIVE: To describe key features and the initial implementation of an employer-sponsored program designed to increase access to quality mental health treatment for employees of a large health care system. Methods: Retrospective data were collected on employer's efforts to develop a programmatic solution to address barriers to accessing quality mental health treatment among its employees and on initial program implementation. Results: Data from the initial cohort ( N = 1049) of program participants support the use of low threshold digital tools to enhance access to care, the importance of care navigation and a robust curated provider network in matching employees to appropriate care options, and the value of providing online, evidence-based psychotherapy to facilitate high rates of treatment engagement. Conclusions: Findings can help inform employers about approaches to improve access to quality mental health treatment for their employees.

Recruitment into Academic Neurosurgery Using a Model for Successful Cross-Campus Research Collaboration: A Premedical Student Survey.

OBJECTIVE: Recruitment of diverse and talented students to the field of neurosurgery is key to its continued growth and scientific advancement. Barriers, including poor perceptions and lack of early exposure, can impact recruitment and have been compounded by the ongoing COVID-19 pandemic. This study examines the impact of an inaugural Neurosurgery Research Consortium meeting on premedical students, assessing whether this exposure generated interest and improved perceptions of a career in neurosurgery. METHODS: Premedical students were recruited to virtually attend an inaugural Neurosurgery Research Consortium developed by the affiliated medical school's American Association of Neurological Surgeons (AANS) Student Chapter. Questionnaires were distributed to students before and after the meeting to assess student demographics and perceptions of neurosurgery. RESULTS: A total of 54 students attended the meeting, with general interest in neurosurgery, medicine, and research opportunities being the primary factors for attendance. Following the research meeting, we found that students perceived neurosurgeons to be friendlier and more approachable, with a more positive quality of life (QoL). Overall perceptions of neurosurgery improved after the meeting, but perceptions among racial and ethnic minority students did not significantly change in the areas of diversity, inclusion, and equity. CONCLUSIONS: These results suggest recruitment strategies targeting undergraduate students may improve their perception of neurosurgery as a career, and may mitigate some barriers to entry. These strategies are cost effective and easily replicable, making an easily implementable approach to provide direct insight into neurosurgery for future medical students while also promoting academic efforts in the field of neurosurgery.

Frailty in Patients Undergoing Surgery for Brain Tumors: A Systematic Review of the Literature.

BACKGROUND: Emerging literature suggests that frailty may be an important driver of postoperative outcomes in patients undergoing surgery for brain tumors. We systematically reviewed the literature on frailty in patients with brain tumor with respect to 3 questions: What methods of frailty assessment have been applied to patients with brain tumor? What thresholds have been defined to distinguish between different levels of frailty? What clinical outcomes does frailty predict in patients with brain tumor? METHODS: A literature search was conducted using PubMed, Embase, The Cochrane Library, Web of Science, Scopus, and ClinicalTrials.gov. Included studies were specific to patients with brain tumor, used a validated instrument to assess frailty, and measured the impact of frailty on postoperative outcomes. RESULTS: Of 753 citations, 21 studies met our inclusion criteria. Frailty instruments were studied, in order of frequency reported, including the 5-factor modified frailty index, 11-factor modified frailty index, Johns Hopkins Adjusted Clinical Groups frailty-defining diagnosis indicator, and Hopkins Frailty Score. Multiple different conventions and thresholds were reported for distinguishing the levels of frailty. Clinical outcomes associated with frailty included mortality, survival, complications, length of stay, charges, costs, discharge disposition, readmissions, and operative time. CONCLUSIONS: Frailty is an increasingly popular concept in patients with brain tumor that is associated with important clinical outcomes. However, the extant literature is largely comprised of retrospective studies with heterogeneous definitions of frailty, thresholds for defining levels of frailty, and patient populations. Further work is needed to understand best practices in assessing frailty in patients with brain tumor and applying these concepts to clinical practice.

Machine Learning-Driven Clinical Image Analysis to Identify Craniosynostosis: A Pilot Study of Telemedicine and Clinic Patients.

BACKGROUND: The authors have developed pretrained machine learning (ML) models to evaluate neonatal head shape deformities using top-down and facial orthogonal photographs of the patient's head. In previous preliminary analysis, this approach was tested with images from an open-source data bank. OBJECTIVE: To determine the accuracy of pretrained ML models in identifying craniosynostosis among patients seen in our outpatient neurosurgery clinic. METHODS: We retrospectively reviewed top-down and facial orthogonal images of each patient's head and provider clinical diagnosis from the same encounters. Head shape classifications generated from 3 pretrained ML models (random forest, classification and regression tree, and linear discriminant analysis) were applied to each patient's photograph data set after craniometric extraction using a predefined image processing algorithm. Diagnoses were codified into a binary scheme of craniosynostosis vs noncraniosynostosis. Sensitivity, specificity, and Matthew correlation coefficient were calculated for software vs provider classifications. RESULTS: A total of 174 patients seen for abnormal head shape between May 2020 and February 2021 were included in the analysis. One hundred seven patients (61%) were seen in-person and 67 (39%) through telemedicine. Twenty-three patients (13%) were diagnosed with craniosynostosis. The best-performing model identified craniosynostosis with an accuracy of 94.8% (95% CI 90.4-97.6), sensitivity of 87.0% (95% CI 66.4-97.2), specificity of 96.0% (95% CI 91.6-98.5), and Matthew correlation coefficient of 0.788 (95% CI 0.725-0.839). CONCLUSION: Machine learning-driven image analysis represents a promising strategy for the identification of craniosynostosis in a real-world practice setting. This approach has potential to reduce the need for imaging and facilitate referral by primary care providers.

Optimizing the residency application process: insights from neurological surgery during the pandemic virtual application cycle.

OBJECTIVE: In this article, the authors describe the impact of the COVID-19 virtual match cycle and discuss approaches to optimize future cycles through applicant and neurosurgical education leadership insights. METHODS: Anonymous surveys of neurosurgery program leaders (program directors and program chairs), program administrators (PAs), and 2020-2021 neurosurgery residency match applicants were distributed by the SNS, in conjunction with the Association of Resident Administrators in Neurological Surgery and AANS Young Neurosurgeons Committee. RESULTS: Responses were received from 77 (67.0%) of 115 PAs, 119 (51.7%) of 230 program leaders, and 124 (44.3%) of 280 applicants representing geographically diverse regions. During the virtual application cycle relative to the previous year, programs received more Electronic Residency Application Service applications (mean 314.8 vs 285.3, p < 0.0001) and conducted more applicant interviews (mean 45.2 vs 39.9, p = 0.0003). More than 50% of applicants applied to > 80 programs; 60.3% received ≤ 20 interview invitations, and 9% received > 40 invitations. Overall, 65% of applicants completed ≤ 20 interviews, whereas 34.7% completed > 20 interviews. Program leaders described one 4-week home subinternship (93.3%) and two 4-week external subinternships (68.9%) as optimal neurosurgical exposure; 62.8% of program leaders found the standardized letter of recommendation template to be somewhat (47.5%) or significantly (15.3%) helpful. Applicants, PAs, and program leaders all strongly preferred a hybrid model of in-person and virtual interview options for future application cycles over all in-person or all virtual options. Ninety-three percent of applicants reported matching within their top 10-ranked programs, and 52.9% of programs matched residents within the same decile ranking as in previous years. CONCLUSIONS: Optimizing a national strategy for the neurosurgery application process that prioritizes equity and reduces costs, while ensuring adequate exposure for applicants to gain educational opportunities and evaluate programs, is critical to maintain a successful training system.

Perceptions of the Virtual Neurosurgery Application Cycle During the Coronavirus Disease 2019 (COVID-19) Pandemic: A Program Director Survey.

OBJECTIVE: The novel coronavirus disease 2019 (COVID-19) pandemic has led to a shift to virtual residency interviews for the 2020-2021 neurosurgery match, with unknown implications for stakeholders. This study seeks to analyze the perceptions of residency program directors (PDs) and associate program directors (APDs) regarding the current virtual format used for residency selection and interviews. METHODS: An anonymous, 30-question survey was constructed and sent to 115 neurosurgery PDs and 26 APDs to assess respondent demographics, factors used to review applicants, perceptions of applicants and applicant engagement, perceptions of standardized letters and interview questions, the effect of the virtual interview format on various stakeholders, and the future outlook for the virtual residency interview format. RESULTS: A total of 38 PDs and APDs completed this survey, constituting a response rate of 27.0%. Survey respondents received significantly more Electronic Residency Application Service applications in the 2020-2021 cycle compared with the 2019-2020 cycle (P = 0.0029). Subinternship performance by home-rotators, (26.3%), letters of recommendation (23.7%), and Step 1 score (18.4%) were ranked as the most important factors for evaluating candidates during the current virtual application cycle. CONCLUSIONS: Our study highlights that applicants applied to a greater number of residency programs compared with years prior, that the criteria used by PDs/APDs to evaluate applicants remained largely consistent compared to previous years, and that the virtual residency interview format may disproportionately disadvantage Doctor of Osteopathic medicine and international medical graduate applicants. Further exploring attitudes toward signaling mechanisms and standardized letters may serve to inform changes to future neurosurgery match cycles.

Actinin BioID reveals sarcomere crosstalk with oxidative metabolism through interactions with IGF2BP2.

Actinins are strain-sensing actin cross-linkers that are ubiquitously expressed and harbor mutations in human diseases. We utilize CRISPR, pluripotent stem cells, and BioID to study actinin interactomes in human cardiomyocytes. We identify 324 actinin proximity partners, including those that are dependent on sarcomere assembly. We confirm 19 known interactors and identify a network of RNA-binding proteins, including those with RNA localization functions. In vivo and biochemical interaction studies support that IGF2BP2 localizes electron transport chain transcripts to actinin neighborhoods through interactions between its K homology (KH) domain and actinin's rod domain. We combine alanine scanning mutagenesis and metabolic assays to disrupt and functionally interrogate actinin-IGF2BP2 interactions, which reveal an essential role in metabolic responses to pathological sarcomere activation using a hypertrophic cardiomyopathy model. This study expands our functional knowledge of actinin, uncovers sarcomere interaction partners, and reveals sarcomere crosstalk with IGF2BP2 for metabolic adaptation relevant to human disease.

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies.

CDK4/6 Dependence of Cyclin D1-Driven Parathyroid Neoplasia in Transgenic Mice.

The protein product of the cyclin D1 oncogene functions by activating partner cyclin-dependent kinases (cdk)4 or cdk6 to phosphorylate, thereby inactivating, the retinoblastoma protein pRB. Nonclassical, cdk-independent, functions of cyclin D1 have been described but their role in cyclin D1-driven neoplasia, with attendant implications for recently approved cdk4/6 chemotherapeutic inhibitors, requires further examination. We investigated whether cyclin D1's role in parathyroid tumorigenesis in vivo is effected primarily through kinase-dependent or kinase-independent mechanisms. Using a mouse model of cyclin D1-driven parathyroid tumorigenesis (PTH-D1), we generated new transgenic lines harboring a mutant cyclin D1 (KE) that is unable to activate its partner kinases. While this kinase-dead KE mutant effectively drove mammary tumorigenesis in an analogous model, parathyroid-overexpressed cyclin D1 KE mice did not develop the characteristic biochemical hyperparathyroidism or parathyroid hypercellularity of PTH-D1 mice. These results strongly suggest that in parathyroid cells, cyclin D1 drives tumorigenesis predominantly through cdk-dependent mechanisms, in marked contrast with the cdk-independence of cyclin D1-driven mouse mammary cancer. These findings highlight crucial tissue-specific mechanistic differences in cyclin D1-driven tumorigenesis, suggest that parathyroid/endocrine cells may be more tumorigenically vulnerable to acquired genetic perturbations in cdk-mediated proliferative control than other tissues, and carry important considerations for therapeutic intervention.

A Contraction Stress Model of Hypertrophic Cardiomyopathy due to Sarcomere Mutations.

Thick-filament sarcomere mutations are a common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered four isogenic induced pluripotent stem cell (iPSC) models of ß-myosin heavy chain and myosin-binding protein C3 mutations, and studied iPSC-derived cardiomyocytes in cardiac microtissue assays that resemble cardiac architecture and biomechanics. All HCM mutations resulted in hypercontractility with prolonged relaxation kinetics in proportion to mutation pathogenicity, but not changes in calcium handling. RNA sequencing and expression studies of HCM models identified p53 activation, oxidative stress, and cytotoxicity induced by metabolic stress that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as a direct consequence of thick-filament mutations, irrespective of mutation localization, and the p53 pathway as a molecular marker of contraction stress and candidate therapeutic target for HCM patients.

Colpocleisis: A Survey of Current Practice Patterns.

BACKGROUND: Currently, there are no standard treatment guidelines for colpocleisis. Clinical practice varies widely for this safe and effective procedure. OBJECTIVE: The aim of this study was to evaluate the current practice patterns in the United States among surgeons who perform colpocleisis. METHODS: A 27-item anonymous Web-based survey was sent to all practicing physicians affiliated with the American Urogynecologic Society. It consisted of questions regarding the demographic background of the physicians and their current practice as it relates to colpocleisis. RESULTS: Of the 1422 physicians contacted, 322 responded (23%) to the questionnaire. Slightly more than half were female with an average time of 15 years in practice. The majority of respondents (79%) were urogynecologists. Most surgeons chose colpocleisis for its high success rate, short operating time, and low risk of complications. Approximately half of the providers performed both LeFort and total colpocleisis. Only 18% performed a routine hysterectomy at the time of surgery. Routine preoperative endometrial evaluation was preferred by 68% of the respondents, with 81% utilizing a transvaginal ultrasound first. Almost all providers would perform concomitant incontinence procedures, with 54% requiring a positive cough stress test and normal postvoid residual. CONCLUSIONS: There is variation in the current practice of colpocleisis in the United States. LeFort colpocleisis is most commonly performed, and routine hysterectomy is uncommon. Two thirds of surgeons evaluate the endometrium prior to surgery. Concomitant anti-incontinence procedures appear to be standard.

Mutational Analysis of ZFY in Sporadic Parathyroid Adenomas.

CONTEXT: The molecular pathogenesis of sporadic parathyroid adenomas is incompletely understood, with alterations in cyclin D1/PRAD1 and MEN1 most firmly established as genetic drivers. The gene encoding the X-linked zinc finger protein (ZFX) has recently been implicated in the pathogenesis of a subset of parathyroid adenomas after recurrent, hotspot-focused somatic mutations were identified. ZFX escapes X inactivation and is transcribed from both alleles in women, and a highly homologous gene encoding the Y-linked zinc finger protein (ZFY) provides dosage compensation in males. OBJECTIVE: We sought to investigate the role of ZFY mutation in sporadic parathyroid adenoma. INTERVENTION: Polymerase chain reaction and Sanger sequencing were used to examine DNA from typically presenting, sporadic (nonfamilial, nonsyndromic) parathyroid adenomas from male patients for mutations within the ZFY gene. RESULTS: No mutations were identified among 117 adenomas. CONCLUSIONS: The absence of ZFY mutations in this series suggests that ZFY rarely, if ever, acts as a driver oncogene in sporadic parathyroid adenomas. The apparent differences in tumorigenic capabilities between the closely related zinc finger proteins ZFX and ZFY suggest that structure-function studies could represent an opportunity to gain insight into neoplastic processes in the parathyroid glands.

A chimeric mechanism for polyvalent trans-phosphorylation of PKA by PDK1.

Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in-trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C-tail of the substrate kinase onto the N-lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1-interacting sites, we discovered a second AGC-conserved motif in the C-tail that interacts with PDK1. Since this motif [FD(X)(1-2)Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1-interacting site in Akt and PRK2, and we predict it will be a PDK1-interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation "switch" that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C-tail serves as a polyvalent element that trans-regulates PDK1 for catalysis. Modeling of the PKA C-tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C-helix, which is positioned by the HF motif. Together, they demonstrate substrate-assisted catalysis involving two kinases that have co-evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C-tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.