ABSTRACT
A critical challenge in genetic diagnostics is the computational assessment of candidate splice variants, specifically the interpretation of nucleotide changes located outside of the highly conserved dinucleotide sequences at the 5' and 3' ends of introns. To address this gap, we developed the Super Quick Information-content Random-forest Learning of Splice variants (SQUIRLS) algorithm. SQUIRLS generates a small set of interpretable features for machine learning by calculating the information-content of wild-type and variant sequences of canonical and cryptic splice sites, assessing changes in candidate splicing regulatory sequences, and incorporating characteristics of the sequence such as exon length, disruptions of the AG exclusion zone, and conservation. We curated a comprehensive collection of disease-associated splice-altering variants at positions outside of the highly conserved AG/GT dinucleotides at the termini of introns. SQUIRLS trains two random-forest classifiers for the donor and for the acceptor and combines their outputs by logistic regression to yield a final score. We show that SQUIRLS transcends previous state-of-the-art accuracy in classifying splice variants as assessed by rank analysis in simulated exomes, and is significantly faster than competing methods. SQUIRLS provides tabular output files for incorporation into diagnostic pipelines for exome and genome analysis, as well as visualizations that contextualize predicted effects of variants on splicing to make it easier to interpret splice variants in diagnostic settings.
Subject(s)
Algorithms , Data Curation/methods , Genetic Diseases, Inborn/genetics , RNA Splice Sites , RNA Splicing , Software , Base Sequence , Computational Biology/methods , Exome , Exons , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/pathology , High-Throughput Nucleotide Sequencing , Humans , Introns , Mutation , Exome SequencingABSTRACT
Human Phenotype Ontology (HPO)-based analysis has become standard for genomic diagnostics of rare diseases. Current algorithms use a variety of semantic and statistical approaches to prioritize the typically long lists of genes with candidate pathogenic variants. These algorithms do not provide robust estimates of the strength of the predictions beyond the placement in a ranked list, nor do they provide measures of how much any individual phenotypic observation has contributed to the prioritization result. However, given that the overall success rate of genomic diagnostics is only around 25%-50% or less in many cohorts, a good ranking cannot be taken to imply that the gene or disease at rank one is necessarily a good candidate. Here, we present an approach to genomic diagnostics that exploits the likelihood ratio (LR) framework to provide an estimate of (1) the posttest probability of candidate diagnoses, (2) the LR for each observed HPO phenotype, and (3) the predicted pathogenicity of observed genotypes. LIkelihood Ratio Interpretation of Clinical AbnormaLities (LIRICAL) placed the correct diagnosis within the first three ranks in 92.9% of 384 case reports comprising 262 Mendelian diseases, and the correct diagnosis had a mean posttest probability of 67.3%. Simulations show that LIRICAL is robust to many typically encountered forms of genomic and phenomic noise. In summary, LIRICAL provides accurate, clinically interpretable results for phenotype-driven genomic diagnostics.
Subject(s)
Computational Biology , Databases, Genetic , Genomics , Rare Diseases/diagnosis , Algorithms , Exome/genetics , Humans , Phenotype , Rare Diseases/genetics , SoftwareABSTRACT
The Human Phenotype Ontology (HPO, https://hpo.jax.org) was launched in 2008 to provide a comprehensive logical standard to describe and computationally analyze phenotypic abnormalities found in human disease. The HPO is now a worldwide standard for phenotype exchange. The HPO has grown steadily since its inception due to considerable contributions from clinical experts and researchers from a diverse range of disciplines. Here, we present recent major extensions of the HPO for neurology, nephrology, immunology, pulmonology, newborn screening, and other areas. For example, the seizure subontology now reflects the International League Against Epilepsy (ILAE) guidelines and these enhancements have already shown clinical validity. We present new efforts to harmonize computational definitions of phenotypic abnormalities across the HPO and multiple phenotype ontologies used for animal models of disease. These efforts will benefit software such as Exomiser by improving the accuracy and scope of cross-species phenotype matching. The computational modeling strategy used by the HPO to define disease entities and phenotypic features and distinguish between them is explained in detail.We also report on recent efforts to translate the HPO into indigenous languages. Finally, we summarize recent advances in the use of HPO in electronic health record systems.
Subject(s)
Biological Ontologies , Computational Biology/methods , Databases, Factual , Disease/genetics , Genome , Phenotype , Software , Animals , Disease Models, Animal , Genotype , Humans , Infant, Newborn , International Cooperation , Internet , Neonatal Screening/methods , Pharmacogenetics/methods , Terminology as TopicABSTRACT
Technological advances in both genome sequencing and prenatal imaging are increasing our ability to accurately recognize and diagnose Mendelian conditions prenatally. Phenotype-driven early genetic diagnosis of fetal genetic disease can help to strategize treatment options and clinical preventive measures during the perinatal period, to plan in utero therapies, and to inform parental decision-making. Fetal phenotypes of genetic diseases are often unique and at present are not well understood; more comprehensive knowledge about prenatal phenotypes and computational resources have an enormous potential to improve diagnostics and translational research. The Human Phenotype Ontology (HPO) has been widely used to support diagnostics and translational research in human genetics. To better support prenatal usage, the HPO consortium conducted a series of workshops with a group of domain experts in a variety of medical specialties, diagnostic techniques, as well as diseases and phenotypes related to prenatal medicine, including perinatal pathology, musculoskeletal anomalies, neurology, medical genetics, hydrops fetalis, craniofacial malformations, cardiology, neonatal-perinatal medicine, fetal medicine, placental pathology, prenatal imaging, and bioinformatics. We expanded the representation of prenatal phenotypes in HPO by adding 95 new phenotype terms under the Abnormality of prenatal development or birth (HP:0001197) grouping term, and revised definitions, synonyms, and disease annotations for most of the 152 terms that existed before the beginning of this effort. The expansion of prenatal phenotypes in HPO will support phenotype-driven prenatal exome and genome sequencing for precision genetic diagnostics of rare diseases to support prenatal care.
Subject(s)
Computational Biology , Placenta , Infant, Newborn , Humans , Female , Pregnancy , Computational Biology/methods , Phenotype , Rare Diseases , Exome SequencingABSTRACT
The Global Alliance for Genomics and Health (GA4GH) Phenopacket Schema was released in 2022 and approved by ISO as a standard for sharing clinical and genomic information about an individual, including phenotypic descriptions, numerical measurements, genetic information, diagnoses, and treatments. A phenopacket can be used as an input file for software that supports phenotype-driven genomic diagnostics and for algorithms that facilitate patient classification and stratification for identifying new diseases and treatments. There has been a great need for a collection of phenopackets to test software pipelines and algorithms. Here, we present phenopacket-store. Version 0.1.12 of phenopacket-store includes 4916 phenopackets representing 277 Mendelian and chromosomal diseases associated with 236 genes, and 2872 unique pathogenic alleles curated from 605 different publications. This represents the first large-scale collection of case-level, standardized phenotypic information derived from case reports in the literature with detailed descriptions of the clinical data and will be useful for many purposes, including the development and testing of software for prioritizing genes and diseases in diagnostic genomics, machine learning analysis of clinical phenotype data, patient stratification, and genotype-phenotype correlations. This corpus also provides best-practice examples for curating literature-derived data using the GA4GH Phenopacket Schema.
ABSTRACT
A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations.
Subject(s)
Amides/chemistry , Small Molecule Libraries/chemistry , Amides/toxicity , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Humans , Neoplastic Stem Cells/drug effects , Small Molecule Libraries/toxicity , Structure-Activity RelationshipABSTRACT
The Global Alliance for Genomics and Health (GA4GH) is a standards-setting organization that is developing a suite of coordinated standards for genomics. The GA4GH Phenopacket Schema is a standard for sharing disease and phenotype information that characterizes an individual person or biosample. The Phenopacket Schema is flexible and can represent clinical data for any kind of human disease including rare disease, complex disease, and cancer. It also allows consortia or databases to apply additional constraints to ensure uniform data collection for specific goals. We present phenopacket-tools, an open-source Java library and command-line application for construction, conversion, and validation of phenopackets. Phenopacket-tools simplifies construction of phenopackets by providing concise builders, programmatic shortcuts, and predefined building blocks (ontology classes) for concepts such as anatomical organs, age of onset, biospecimen type, and clinical modifiers. Phenopacket-tools can be used to validate the syntax and semantics of phenopackets as well as to assess adherence to additional user-defined requirements. The documentation includes examples showing how to use the Java library and the command-line tool to create and validate phenopackets. We demonstrate how to create, convert, and validate phenopackets using the library or the command-line application. Source code, API documentation, comprehensive user guide and a tutorial can be found at https://github.com/phenopackets/phenopacket-tools. The library can be installed from the public Maven Central artifact repository and the application is available as a standalone archive. The phenopacket-tools library helps developers implement and standardize the collection and exchange of phenotypic and other clinical data for use in phenotype-driven genomic diagnostics, translational research, and precision medicine applications.
Subject(s)
Neoplasms , Software , Humans , Genomics , Databases, Factual , Gene LibraryABSTRACT
Navigating the vast landscape of clinical literature to find optimal treatments and management strategies can be a challenging task, especially for rare diseases. To address this task, we introduce the Medical Action Ontology (MAxO), the first ontology specifically designed to organize medical procedures, therapies, and interventions in a structured way. Currently, MAxO contains 1757 medical action terms added through a combination of manual and semi-automated processes. MAxO was developed with logical structures that make it compatible with several other ontologies within the Open Biological and Biomedical Ontologies (OBO) Foundry. These cover a wide range of biomedical domains, from human anatomy and investigations to the chemical and protein entities involved in biological processes. We have created a database of over 16000 annotations that describe diagnostic modalities for specific phenotypic abnormalities as defined by the Human Phenotype Ontology (HPO). Additionally, 413 annotations are provided for medical actions for 189 rare diseases. We have developed a web application called POET (https://poet.jax.org/) for the community to use to contribute MAxO annotations. MAxO provides a computational representation of treatments and other actions taken for the clinical management of patients. The development of MAxO is closely coupled to the Mondo Disease Ontology (Mondo) and the Human Phenotype Ontology (HPO) and expands the scope of our computational modeling of diseases and phenotypic features to include diagnostics and therapeutic actions. MAxO is available under the open-source CC-BY 4.0 license (https://github.com/monarch-initiative/MAxO).
ABSTRACT
BACKGROUND: Navigating the clinical literature to determine the optimal clinical management for rare diseases presents significant challenges. We introduce the Medical Action Ontology (MAxO), an ontology specifically designed to organize medical procedures, therapies, and interventions. METHODS: MAxO incorporates logical structures that link MAxO terms to numerous other ontologies within the OBO Foundry. Term development involves a blend of manual and semi-automated processes. Additionally, we have generated annotations detailing diagnostic modalities for specific phenotypic abnormalities defined by the Human Phenotype Ontology (HPO). We introduce a web application, POET, that facilitates MAxO annotations for specific medical actions for diseases using the Mondo Disease Ontology. FINDINGS: MAxO encompasses 1,757 terms spanning a wide range of biomedical domains, from human anatomy and investigations to the chemical and protein entities involved in biological processes. These terms annotate phenotypic features associated with specific disease (using HPO and Mondo). Presently, there are over 16,000 MAxO diagnostic annotations that target HPO terms. Through POET, we have created 413 MAxO annotations specifying treatments for 189 rare diseases. CONCLUSIONS: MAxO offers a computational representation of treatments and other actions taken for the clinical management of patients. Its development is closely coupled to Mondo and HPO, broadening the scope of our computational modeling of diseases and phenotypic features. We invite the community to contribute disease annotations using POET (https://poet.jax.org/). MAxO is available under the open-source CC-BY 4.0 license (https://github.com/monarch-initiative/MAxO). FUNDING: NHGRI 1U24HG011449-01A1 and NHGRI 5RM1HG010860-04.
Subject(s)
Biological Ontologies , Humans , Rare Diseases , Software , Computer SimulationABSTRACT
A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).
Subject(s)
Breast Neoplasms/drug therapy , Hydrazones/pharmacology , Neoplastic Stem Cells/cytology , Pyrroles/pharmacology , Breast Neoplasms/pathology , Female , HumansABSTRACT
Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a chi(2) analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, alpha-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that alpha-actinin and densin directly interact with residues 151-300 and 446-817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum.
Subject(s)
Microfilament Proteins/metabolism , Neostriatum/metabolism , Nerve Tissue Proteins/metabolism , Proteomics/methods , Actinin/chemistry , Actinin/metabolism , Aging/metabolism , Amino Acid Sequence , Animals , Cell Line , Gene Knockout Techniques , Humans , Immunoprecipitation , Mass Spectrometry , Mice , Microfilament Proteins/chemistry , Molecular Sequence Data , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/chemistry , Protein Binding , Protein Transport , Rats , Reproducibility of Results , Solubility , Subcellular Fractions/metabolismABSTRACT
Ontologies have emerged to become critical to support data and knowledge representation, standardization, integration, and analysis. The SARS-CoV-2 pandemic led to the rapid proliferation of COVID-19 data, as well as the development of many COVID-19 ontologies. In the interest of supporting data interoperability, we initiated a community-based effort to harmonize COVID-19 ontologies. Our effort involves the collaborative discussion among developers of seven COVID-19 related ontologies, and the merging of four ontologies. This effort demonstrates the feasibility of harmonizing these ontologies in an interoperable framework to support integrative representation and analysis of COVID-19 related data and knowledge.
ABSTRACT
We report the outcome of a high-throughput small-molecule screen to identify novel, nontoxic, inhibitors of Trypansoma cruzi, as potential starting points for therapeutics to treat for both the acute and chronic stages of Chagas disease. Two compounds were identified that displayed nanomolar inhibition of T. cruzi and an absence of activity against host cells at the highest tested dose. These compounds have been registered with NIH Molecular Libraries Program (probes ML157 and ML158).
Subject(s)
Small Molecule Libraries , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Inhibitory Concentration 50 , Molecular Structure , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship , Trypanocidal Agents/chemistryABSTRACT
BACKGROUND: Defects in the glycosylphosphatidylinositol (GPI) biosynthesis pathway can result in a group of congenital disorders of glycosylation known as the inherited GPI deficiencies (IGDs). To date, defects in 22 of the 29 genes in the GPI biosynthesis pathway have been identified in IGDs. The early phase of the biosynthetic pathway assembles the GPI anchor (Synthesis stage) and the late phase transfers the GPI anchor to a nascent peptide in the endoplasmic reticulum (ER) (Transamidase stage), stabilizes the anchor in the ER membrane using fatty acid remodeling and then traffics the GPI-anchored protein to the cell surface (Remodeling stage). RESULTS: We addressed the hypothesis that disease-associated variants in either the Synthesis stage or Transamidase+Remodeling-stage GPI pathway genes have distinct phenotypic spectra. We reviewed clinical data from 58 publications describing 152 individual patients and encoded the phenotypic information using the Human Phenotype Ontology (HPO). We showed statistically significant differences between the Synthesis and Transamidase+Remodeling Groups in the frequencies of phenotypes in the musculoskeletal system, cleft palate, nose phenotypes, and cognitive disability. Finally, we hypothesized that phenotypic defects in the IGDs are likely to be at least partially related to defective GPI anchoring of their target proteins. Twenty-two of one hundred forty-two proteins that receive a GPI anchor are associated with one or more Mendelian diseases and 12 show some phenotypic overlap with the IGDs, represented by 34 HPO terms. Interestingly, GPC3 and GPC6, members of the glypican family of heparan sulfate proteoglycans bound to the plasma membrane through a covalent GPI linkage, are associated with 25 of these phenotypic abnormalities. CONCLUSIONS: IGDs associated with Synthesis and Transamidase+Remodeling stages of the GPI biosynthesis pathway have significantly different phenotypic spectra. GPC2 and GPC6 genes may represent a GPI target of general disruption to the GPI biosynthesis pathway that contributes to the phenotypes of some IGDs.
Subject(s)
Glycosylphosphatidylinositols , Seizures , Aminoacyltransferases , Glycosylphosphatidylinositols/genetics , Glypicans , Humans , Mutation/genetics , PhenotypeABSTRACT
Protein phosphatase 1 (PP1) catalytic subunits dephosphorylate specific substrates in discrete subcellular compartments to modulate many cellular processes. Canonical PP1-binding motifs (R/K-V/I-X-F) in a family of proteins mediate subcellular targeting, and the amino acids that form the binding pocket for the canonical motif are identical in all PP1 isoforms. However, PP1gamma1 but not PP1beta is selectively localized to F-actin-rich dendritic spines in neurons. Although the F-actin-binding proteins neurabin I and spinophilin (neurabin II) also bind PP1, their role in PP1 isoform selective targeting in intact cells is poorly understood. We show here that spinophilin selectively targets PP1gamma1, but not PP1beta, to F-actin-rich cortical regions of intact cells. Mutation of a PP1gamma1 selectivity determinant (N(464)EDYDRR(470) in spinophilin: conserved as residues 473-479 in neurabin) to VKDYDTW severely attenuated PP1gamma1 interactions with neurabins in vitro and in cells and disrupted PP1gamma1 targeting to F-actin. This domain is not involved in the weaker interactions of neurabins with PP1beta. In contrast, mutation of the canonical PP1-binding motif attenuated interactions of neurabins with both isoforms. Thus, selective targeting of PP1gamma1 to F-actin by neurabins in intact cells requires both the canonical PP1-binding motif and an auxiliary PP1gamma1-selectivity determinant.
Subject(s)
Actins/metabolism , Protein Phosphatase 1/metabolism , Amino Acid Sequence , Animals , Binding Sites , Brain/cytology , Brain/metabolism , Catalysis , Cell Line , Humans , Protein Binding , Protein Phosphatase 1/chemistry , Protein Phosphatase 1/genetics , Protein Structure, Tertiary , Protein Subunits , Rats , TransfectionABSTRACT
The Human Phenotype Ontology (HPO) is a standardized set of phenotypic terms that are organized in a hierarchical fashion. It is a widely used resource for capturing human disease phenotypes for computational analysis to support differential diagnostics. The HPO is frequently used to create a set of terms that accurately describe the observed clinical abnormalities of an individual being evaluated for suspected rare genetic disease. This profile is compared with computational disease profiles in the HPO database with the aim of identifying genetic diseases with comparable phenotypic profiles. The computational analysis can be coupled with the analysis of whole-exome or whole-genome sequencing data through applications such as Exomiser. This article explains how to choose an optimal set of HPO terms for these cases and enter them with software, such as PhenoTips and PatientArchive, and demonstrates how to use Phenomizer and Exomiser to generate a computational differential diagnosis. © 2019 by John Wiley & Sons, Inc.
Subject(s)
Biological Ontologies , Computational Biology , Databases, Genetic , Genetic Diseases, Inborn/diagnosis , Software , Diagnosis, Differential , Exome/genetics , Genetic Diseases, Inborn/genetics , Humans , Phenotype , Whole Genome SequencingABSTRACT
Electronic Health Record (EHR) systems typically define laboratory test results using the Laboratory Observation Identifier Names and Codes (LOINC) and can transmit them using Fast Healthcare Interoperability Resource (FHIR) standards. LOINC has not yet been semantically integrated with computational resources for phenotype analysis. Here, we provide a method for mapping LOINC-encoded laboratory test results transmitted in FHIR standards to Human Phenotype Ontology (HPO) terms. We annotated the medical implications of 2923 commonly used laboratory tests with HPO terms. Using these annotations, our software assesses laboratory test results and converts each result into an HPO term. We validated our approach with EHR data from 15,681 patients with respiratory complaints and identified known biomarkers for asthma. Finally, we provide a freely available SMART on FHIR application that can be used within EHR systems. Our approach allows readily available laboratory tests in EHR to be reused for deep phenotyping and exploits the hierarchical structure of HPO to integrate distinct tests that have comparable medical interpretations for association studies.
ABSTRACT
Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.
ABSTRACT
Expression of recombinant PP1 isoforms with fully authentic properties has proven to be a challenge for several laboratories. In order to circumvent this technical limitation in the investigation of isoform-specific roles for PP1, methods have been developed to analyze specific properties of native PP1 isoforms. The well-documented method of ethanol precipitation of tissue extracts has been used to dissociate phosphatase catalytic subunits from their endogenous regulatory subunits and other cellular proteins. Although very low levels of PP1 and PP2A regulatory subunits are sometimes detected in PPC preparations, they are not associated with their respective catalytic subunits because they do not copurify with the catalytic subunits on microcystin-Sepharose (Bauman & Colbran, not shown). Thus, the PPC preparation represents a mixture of native monomeric phosphatase catalytic subunits (including PP1 isoforms, PP2AC, PP4C, and PP6C) that can be used to analyze their interactions with other proteins. The methods described in this report rely on the availability of highly specific antibodies to PP1 isoforms. The sheep antibodies have previously proven effective for immunoblotting and immunoprecipitation, whereas rabbit antibodies have also been used for immunocytochemistry. This paper documents the use of these antibodies in Far-Western overlay and glutathione-agarose cosedimentation assays to investigate interactions of specific PP1 isoforms with recombinant fragments of PP1-targeting subunits (spinophilin, neurabin and GM). Moreover, covalent coupling of affinity-purified sheep antibodies to agarose provided a means for the immuno-isolation of PP1 beta and PP1 gamma 1 from the PPC preparation. Active catalytic subunits are recovered from the affinity resin using chaotropic agents, permitting for the first time the assessment of the effects of specific targeting subunits on activities of individual native PP1 isoforms. These methods have been used successfully to demonstrate that some PP1-interacting proteins discriminate among the isoforms. The isoform inhibition assays provide a measure of the binding equilibrium in the milieu of the phosphatase assay. For example, while some PP1-binding proteins inhibit native PP1 beta and native PP1 gamma 1 with equivalent potency (e.g., PKA-phosphorylated inhibitor-1), spinophilin, neurabin and GM differentiate between these two isoforms; spinophilin and neurabin fragments inhibit native PP1 gamma 1 approximately 20-fold more potently than they inhibit native PP1 beta (Fig. 4), whereas GM inhibits native PP1 beta more potently than native PP1 gamma 1 (not shown). Moreover, the activity of native PP1 gamma 1 is approximately 100-fold more sensitive to neurabin and spinophilin than is the activity of bacterially-expressed recombinant PP1 gamma 1 (Fig. 4). The interpretation of these inhibition assays is consistent with data obtained in Far-Western overlay (Fig. 2) and glutathione-agarose cosedimentation assays (Fig. 3), which assess more stable interactions of PP1 isoforms. Thus, spinophilin and neurabin selectively bind PP1 gamma 1 over PP1 beta, whereas GM is highly selective for PP1 beta. These data are consistent with previous experiments that showed spinophilin and neurabin are present in PP1 gamma 1 complexes in brain extracts, but not in PP1 beta complexes. Moreover, only PP1 beta has been identified in complexes with GM in muscle extracts, although these data did not exclude the possibility that other isoforms were also present. Presumably, these isoform-selective interactions confer different functions on PP1. In summary, we have developed methods that should prove useful in defining the isoform-selectivity of other PP1-targeting subunits. Moreover, these methods may be employed to identify domains in PP1-interacting proteins that confer isoform specificity. Similar strategies may also be used to explore interactions of protein phosphatase catalytic subunits with other proteins.