Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

No abstract

Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions. Action on multiple substrates (substrate ambiguity) can be harnessed for performance of functions in the cell that supersede catalysis of a single metabolite. These functions include proofreading, scavenging of nutrients, removal of antimetabolites, balancing of metabolite pools, and establishing system redundancy. In this review, we present examples of enzymes that perform these cellular roles by leveraging substrate ambiguity and then present the structural features that support both specificity and ambiguity. We focus on the phosphatases of the haloalkanoate dehalogenase superfamily and the thioesterases of the hotdog fold superfamily.In the 1990s, a series of studies on the evolution of catalysis in protein fold families helped define contemporary understanding of enzymes as potentially promiscuous catalysts; the analyses of these enzyme superfamilies suggested that certain folds showed higher variability than expected with regard to the chemistries that can be catalyzed or the substrates that can be acted on (1-11). To summarize, the current model holds that enzyme families grow as a result of gene duplication coupled with the acquisition of an advantageous new function. Because the backbone folds, and thus, the catalytic scaffolds are inherited, so is the chemical trait that underlies the intrinsic catalytic functions of all family members. In enzyme families, evidence can be found for low level intrinsic activity associated with one or more extant members, co-existing with the high level of activity unique to the subject enzyme (see for instance, the enolase and alkaline phosphatase enzyme superfamilies (12, 13)). The ability to carry out such alternate chemistry is termed catalytic promiscuity. The plausible link between catalytic promiscuity and evolvability has been explored in previous publications (for recent coverage and reviews of this topic, see Refs. 14 -17).The most commonly encountered observation of promiscuity involves the catalysis of one type of chemistry with many different substrates. Jensen (18) referred to this trait as "substrate ambiguity," and this is the name we will use. Herein, we examine the selective advantage associated with activity toward multiple substrates by highlighting specific examples of enzymes for which the level of substrate ambiguity runs high to fulfill specific roles in the cell. We use as examples enzymes from the haloalkanoate dehalogenase (HAD) 3 superfamily and the thioesterases of the hotdog fold superfamily. In addition, we dissect the architectures of enzymes from these families to discover underlying structural sources of specificity and substrate ambiguity. Screening to Assess Substrate AmbiguityIn vitro enzyme activity measurements carried out with a structurally diverse library of potential substrates allow one to generate a substrate specificity profile for the enzyme of interest. However, the mo...

BackgroundPersonalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics.MethodsWe developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data.ResultsWe returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases.ConclusionsThese results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0313-0) contains supplementary material, which is available to authorized users.

The explosion of protein sequence information requires that current strategies for function assignment must evolve to complement experimental approaches with computationally-based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown Haloalkanoate Dehalogenase superfamily member BT2127 (Uniprot accession # Q8A5V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics/structure/mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with kcat/Km value for pyrophosphate of ∼1 × 105 M−1 s−1), together with the gene context, supports the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of the wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure guided site directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. Based on this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172 and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue β-phosphoglucomutase control the leaving group size (phosphate vs. glucose-phosphate) and the position of the Asp acid/base in the open vs. closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

Although the universe of protein structures is vast, these innumerable structures can be categorized into a finite number of folds. New functions commonly evolve by elaboration of existing scaffolds, for example, via domain insertions. Thus, understanding structural diversity of a protein fold evolving via domain insertions is a fundamental challenge. The haloalkanoic dehalogenase superfamily serves as an excellent model system wherein a variable cap domain accessorizes the ubiquitous Rossmann-fold core domain. Here, we determine the impact of the cap-domain insertion on the sequence and structure divergence of the core domain. Through quantitative analysis on a unique dataset of 154 core-domain-only and capdomain-only structures, basic principles of their evolution have been uncovered. The relationship between sequence and structure divergence of the core domain is shown to be monotonic and independent of the corresponding type of domain insert, reflecting the robustness of the Rossmann fold to mutation. However, core domains with the same cap type share greater similarity at the sequence and structure levels, suggesting interplay between the cap and core domains. Notably, results reveal that the variance in structure maps to α-helices flanking the central β-sheet and not to the domain-domain interface. Collectively, these results hint at intramolecular coevolution where the fold diverges differentially in the context of an accessory domain, a feature that might also apply to other multidomain superfamilies. directed evolution | phosphoryl transferase | protein evolution | structural bioinformatics | HAD superfamily T he universe of protein structures is vast and diverse, yet these innumerable structures can be categorized into a finite number of folds (1). Ideally, the protein fold has a robust yet evolvable architecture to deliver chemistry, bind interaction partners, or provide scaffolding. A popular strategy for the acquisition of new function(s) is the topological alteration of the fold to provide a new evolutionary platform. More frequently, existing and stable scaffolds are elaborated to attain diversity that is due to accumulation of stochastic, independent, and near-neutral mutations in the protein sequence. In a large number of cases, the expansion of functional space has been achieved by the tandem fusion of two or three domains to form evolutionary modules known as supradomains (2). An analysis of catalytic domains fused to the nucleotide-binding Rossmann domain has revealed that the sequential order of their connections is conserved because each pairing arose from a single recombination event (3). Another common structural embellishment is that of domain insertion(s) into existing folds (4)-a strategy that is ubiquitous in all structural classes, i.e., all α, all β, α + β, and α/β (5). For example, members of the A, B, and Y DNA polymerase superfamilies, Rab geranylgeranyl transferase superfamily, and alcohol dehydrogenase superfamily have inserted different domains into the native fold to fine tun...

Summary Adenoid cystic carcinoma (ACC) is a rare cancer type that originates in the salivary glands. Tumors commonly invade along nerve tracks in the head and neck, making surgery challenging. Follow-up treatments for recurrence or metastasis including chemotherapy and targeted therapies have shown limited efficacy, emphasizing the need for new therapies. Here, we report a Drosophila-based therapeutic approach for a patient with advanced ACC disease. A patient-specific Drosophila transgenic line was developed to model the five major variants associated with the patient's disease. Robotics-based screening identified a three-drug cocktail—vorinostat, pindolol, tofacitinib—that rescued transgene-mediated lethality in the Drosophila patient-specific line. Patient treatment led to a sustained stabilization and a partial metabolic response of 12 months. Subsequent resistance was associated with new genomic amplifications and deletions. Given the lack of options for patients with ACC, our data suggest that this approach may prove useful for identifying novel therapeutic candidates.

Introduction: B-cell maturation antigen (BCMA) is primarily expressed by malignant and normal plasma cells, making it an attractive target for the treatment of multiple myeloma (MM). bb21217 is a BCMA-directed chimeric antigen receptor (CAR) T cell therapy that uses the same CAR molecule as idecabtagene vicleucel (ide-cel, bb2121), but adds the PI3K inhibitor bb007 during manufacturing to enrich the drug product (DP) for memory-like T cells, thereby reducing the proportion of highly differentiated or senescent T cells. We conducted correlative analyses to investigate the mechanistic hypothesis that CAR+ T cells with memory like phenotypes may persist and function longer, which may be one determinant of duration of response (DOR). Methods: An ongoing phase I clinical study (CRB-402; NCT03274219) is assessing safety and efficacy of bb21217 in relapsed/refractory MM patients. A total of 44 patients had PBMCs, collected from apheresis, and DP characterized by RNA sequencing (RNAseq) and mass Cytometry (CyTOF). The correlation of T cell phenotype with peak expansion, response and DOR per IMWG Uniform Response Criteria was explored. P-values were determined by Wilcoxon test, Spearman correlation, or Cox PH regression on DOR with categorical marker values (high/low). Results: In this patient population, substantial cross patient heterogeneity in T cell phenotypes was observed both in PBMCs and DP. Late differentiation/senescent markers in PBMCs were negatively correlated with clinical response. In particular, patients whose DP had higher expression of CD57 had lower peak expansion (p<0.0001), experienced more early relapse by month 6 (M6) (p<0.05) and had lower DOR (p<0.05) compared to those with lower CD57 expression. Paired analysis of PBMCs and DP demonstrated bb21217 DP is enriched for memory-like T cells (LEF-1+ (median increase 310%, p<0.0001), CD27+ (median increase 84.7%, p<0.0001), CCR7+ (median increase 188.3%, p<0.0001) and depleted of highly differentiated or senescent CD57+ T cells (median decrease 87.3%, p<0.0001), relative to PBMCs. As CAR+ T cell peak expansion is associated with initial clinical response, we investigated the relationship between peak expansion and DP phenotype. Expression of early memory T cell markers (eg, LEF1, CD27, CCR7) in DP were positively correlated with peak expansion, while markers of terminally differentiated effector cell markers in DP (eg, CD57, GZMA, GZMB) were negatively correlated with peak expansion. RNAseq also showed a significant enrichment in naïve/early memory gene signatures and a decrease in late differentiation gene signatures in DP from patients with high peak expansion (>2x105copies/ug), consistent with the CYTOF findings. Early activation markers (CD38 p<0.001, IRF4 p<0.01) were expressed in the DP from patients with high peak expansion, suggesting that an early activation phenotype may contribute to robust expansion. We assessed the relationship between T cell memory markers in DP and sustained response by comparing DP from patients with or without progressive disease (PD) by M6. RNAseq analysis showed a significant enrichment for early memory (CCR7, p<0.05, SELL, p<0.05) and early activation (CD38 p<0.05, IRF4 p<0.05) T cell phenotypes and a significant reduction in late differentiation/senescence (CD57 p<0.05, GZMB p<0.01. KLRG1 p<0.01) T cell phenotypes in DP from patients without PD at M6. Gene set enrichment analysis showed enrichment of naïve/memory gene signatures in patients without PD at M6. ScRNAseq analyses from a subset of PBMC/DP as well as immunophenotyping data from clinical samples, including memory phenotypes, and their relationship to clinical outcome, will also be presented. Conclusion: As seen with other CAR T cell studies, the quality of incoming PBMCs, in particular the fraction of T cells with a late differentiation/senescent phenotype, influences initial and sustained clinical response. The analyses reported here support the mechanistic hypothesis of bb21217, suggesting the presence of early memory like T cells in PBMC and/or DP may contribute to high peak expansion and prolonged DOR, while presence of highly differentiated or senescent T cells may negatively impact these measures. Further clinical evaluation of bb21217 and robust correlative analyses will be important to help contextualize the influence of patient and product characteristics on clinical outcomes. Disclosures Finney: bluebird bio: Current Employment, Current equity holder in publicly-traded company. Yeri:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Mao:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Pandya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Alonzo:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hopkins:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hymson:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hu:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Foos:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bhadoriya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hintzen:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Gioia:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Timm:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Massaro:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hause:Bristol-Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Kaiser:BMS: Current Employment, Current equity holder in publicly-traded company. Martin:BMS: Current Employment, Current equity holder in publicly-traded company. Shah:BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding; GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy. Raje:Caribou: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Amgen: Consultancy. Berdeja:Novartis: Research Funding; Lilly: Research Funding; Legend: Consultancy; Takeda: Consultancy, Research Funding; Servier: Consultancy; Teva: Research Funding; Vivolux: Research Funding; Bluebird: Research Funding; Acetylon: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Research Funding; BMS: Consultancy, Research Funding; Bioclinica: Consultancy; CRISPR Therapeutics: Consultancy, Research Funding; Constellation: Research Funding; Cellularity: Research Funding; Celgene: Consultancy, Research Funding; Glenmark: Research Funding; Genentech, Inc.: Research Funding; EMD Sorono: Research Funding; CURIS: Research Funding; Kite Pharma: Consultancy; Kesios: Research Funding; Karyopharm: Consultancy; Janssen: Consultancy, Research Funding; Prothena: Consultancy; Poseida: Research Funding. Grande:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bitter:Novartis AG, Predicant Biosciences, Biospect, F Hofmann-La Roche: Ended employment in the past 24 months; bluebird bio: Current Employment, Current equity holder in publicly-traded company; Novartis: Ended employment in the past 24 months, Patents & Royalties. Petrocca:bluebird, bio: Current Employment, Current equity holder in publicly-traded company. Friedman:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Sangurdekar:bluebird bio: Current Employment, Current equity holder in publicly-traded company; Biogen: Ended employment in the past 24 months.