Joseph Paggi

All Publications

• Structure Insights into Biased Signaling of kappa Opioid Receptor. FASEB journal : official publication of the Federation of American Societies for Experimental Biology El Daibani, A., Paggi, J., Kim, K., Laloudakis, Y., Popov, P., Bernhard, S., Olsen, R. H., Diberto, J., Katritch, V., Wunsch, B., Dror, R., Che, T. 2022; 36 Suppl 1

  Abstract

  kappa-opioid receptor (KOR) has been known as an attractive drug target for pain management because it offers anti-nociception without causing constipation, euphoria, or respiratory depression that accompany traditional analgesics targeting the mu-opioid receptor (MOR). Although negative side-effects of KOR activation prevent the adoption of most KOR-based analgesics such as hallucinations, dysphoria, and sedation, nalfurafine is the only clinically available KOR agonist that shows atypical properties relative to other KOR agonists such as no hallucination and no dysphoria. To better understand the molecular determinants that drive KOR's therapeutic property, here we reported the crystal structure of KOR bound to nalfurafine and identified the structural features that contribute to nalfurafine's high potency and unique signaling profile. Using atomic-level molecular dynamics (MD) simulations of KOR bound to G protein-biased nalfurafine and other differently biased ligands, we identified receptor conformations that cause nalfurafine's favorable therapeutic profile and provide a hypothesis for the structural basis for signaling bias at KOR in general.

  View details for DOI 10.1096/fasebj.2022.36.S1.L8027

  View details for PubMedID 35555267

• S-CAP extends pathogenicity prediction to genetic variants that affect RNA splicing. Nature genetics Jagadeesh, K. A., Paggi, J. M., Ye, J. S., Stenson, P. D., Cooper, D. N., Bernstein, J. A., Bejerano, G. 2019

  Abstract

  Exome analysis of patients with a likely monogenic disease does not identify a causal variant in over half of cases. Splice-disrupting mutations make up the second largest class of known disease-causing mutations. Each individual (singleton) exome harbors over 500 rare variants of unknown significance (VUS) in the splicing region. The existing relevant pathogenicity prediction tools tackle all non-coding variants as one amorphic class and/or are not calibrated for the high sensitivity required for clinical use. Here we calibrate seven such tools and devise a novel tool called Splicing Clinically Applicable Pathogenicity prediction (S-CAP) that is over twice as powerful as all previous tools, removing 41% of patient VUS at 95% sensitivity. We show that S-CAP does this by using its own features and not via meta-prediction over previous tools, and that splicing pathogenicity prediction is distinct from predicting molecular splicing changes. S-CAP is an important step on the path to deriving non-coding causal diagnoses.

  View details for PubMedID 30804562

• A sequence-based, deep learning model accurately predicts RNA splicing branchpoints. RNA (New York, N.Y.) Paggi, J. M., Bejerano, G. 2018

  Abstract

  Experimental detection of RNA splicing branchpoints is difficult. To date, high-confidence experimental annotations exist for 18% of 3' splice sites in the human genome. We develop a deep-learning based branchpoint predictor, LaBranchoR, which predicts a correct branchpoint for at least 75% of 3' splice sites genome-wide. Detailed analysis of cases in which our predicted branchpoint deviates from experimental data suggests a correct branchpoint is predicted in over 90% of cases. We use our predicted branchpoints to identify a novel sequence element upstream of branchpoints consistent with extended U2 snRNA base pairing, show an association between weak branchpoints and alternative splicing, and explore the effects of genetic variants on branchpoints. We provide genome-wide branchpoint annotations and in silico mutagenesis scores at http://bejerano.stanford.edu/labranchor.

  View details for PubMedID 30224349

• Structural mechanisms of selectivity and gating in anion channelrhodopsins. Nature Kato, H. E., Kim, Y. S., Paggi, J. M., Evans, K. E., Allen, W. E., Richardson, C., Inoue, K., Ito, S., Ramakrishnan, C., Fenno, L. E., Yamashita, K., Hilger, D., Lee, S. Y., Berndt, A., Shen, K., Kandori, H., Dror, R. O., Kobilka, B. K., Deisseroth, K. 2018

  Abstract

  Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anionchannels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

  View details for PubMedID 30158697

• Crystal structure of the natural anion-conducting channelrhodopsin GtACR1. Nature Kim, Y. S., Kato, H. E., Yamashita, K., Ito, S., Inoue, K., Ramakrishnan, C., Fenno, L. E., Evans, K. E., Paggi, J. M., Dror, R. O., Kandori, H., Kobilka, B. K., Deisseroth, K. 2018

  Abstract

  The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anionconductance and high anionselectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9A resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

  View details for PubMedID 30158696

• Numerous recursive sites contribute to accuracy of splicing in long introns in flies PLOS GENETICS Pai, A. A., Paggi, J. M., Yan, P., Adelman, K., Burge, C. B. 2018; 14 (8): e1007588

  Abstract

  Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.

  View details for PubMedID 30148878

• Structure of the µ-opioid receptor-Gi protein complex. Nature Koehl, A., Hu, H., Maeda, S., Zhang, Y., Qu, Q., Paggi, J. M., Latorraca, N. R., Hilger, D., Dawson, R., Matile, H., Schertler, G. F., Granier, S., Weis, W. I., Dror, R. O., Manglik, A., Skiniotis, G., Kobilka, B. K. 2018

  Abstract

  The mu-opioid receptor (muOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by muOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5A resolution cryo-electron microscopy structure of the muOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its Nterminus interacting with conserved receptor residues and its Cterminus engaging regions important for opioid-ligand selectivity. Comparison of the muOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein alpha-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the OR.

  View details for PubMedID 29899455