Structural and functional analysis of parameters governing tankyrase-1 interaction with telomeric repeat-binding factor 1 and GDP-mannose 4,6-dehydratase.

Human tankyrase-1 (TNKS) is a member of the poly(ADPribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: The PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity

Structural and functional analysis of parameters governing tankyrase-1 interaction with telomeric repeat-binding factor 1 and GDP-mannose 4,6-dehydratase.

Human tankyrase-1 (TNKS) is a member of the poly(ADPribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: The PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity

Making your own Penicillin after the Zombie Apocalypse

Presentation: 47:5

Structural Analysis of Tankyrase-1 Regulation and Binding Partner Interaction

Poly(ADP-Ribose) (PAR) is a transient posttranslational modification that is involved in the regulation of several critical cellular processes including DNA damage repair and Wnt signaling. PAR is synthesized by Poly(ADP-Ribose) Polymerases (PARPs), that utilize NAD+ as a substrate to modify themselves and target proteins with PAR (termed PARylation). PARPs possess conserved catalytic PARP (CAT) domains, however the unique regulatory domains of individual family members endow each PARP with their own unique function(s). Much is understood about the regulatory mechanism of founding family member PARP-1. However, although several biological functions have been identified for other family members, a gap persists in our understanding of their regulatory mechanisms. Tankyrase-1 (TNKS, or PARP-5A) interacts with a growing number of structurally and functionally unique binding partners utilizing a multivalent ankyrin repeat domain. Through these interactions TNKS regulates several critical processes including Wnt signaling, Golgi trafficking, telomere maintenance, and apoptosis, and inhibition of TNKS catalytic activity has applications in the treatment of fibrosis as well as colorectal and gastric cancer. Although binding is required for PARylation, some binding partners can engage TNKS without being modified. TNKS is therefore capable of promiscuous binding while simultaneously deciding which partners do and do not get PARylated. Unfortunately, binding partners demonstrate such remarkable heterogeneity that no pattern has yet emerged that could identify a unifying regulatory mechanism. Prior to the work presented here, the study of the ankyrin repeat domain had been restricted to small fragments, and only limited studies had been performed that analyzed TNKS interaction with full length binding partners. To better understand the factors that govern TNKS regulation, we utilized a multifaceted structural and biochemical approach to define TNKS as a binding platform, and elucidate the features of TNKS:binding partner interactions that are required for binding and PARylation. This study has provided the first structural analysis of the entire ankyrin repeat domain, as well as the first comprehensive analysis of TNKS interaction with full-length binding partners. Our data demonstrate that the ankyrin repeat domain is a heteromultivalent binding platform that can dynamically sample unique conformations in order to facilitate binding. Although this domain accommodates significant variation amongst its many binding partners, PARylated binding partner telomeric repeat binding factor 1 (TRF1) possesses unique sequence and structural features that are critical for TNKS interaction and PARylation. However, we also found that the same factors do not affect the mechanism that prevents modification of non-PARylated binding partner GDP-Mannose-4,6-Dehydratase (GMD), indicating that there are additional factors that can affect TNKS regulation. Our data thus highlight the need for further study of the TNKS:binding partner interface in order to identify additional parameters that govern interaction, and elucidate the mechanisms that drive TNKS regulation. Importantly, this study provides unique insights into the characteristics of the ankyrin repeat domain that allow TNKS to function as a master scaffolding protein, and regulate multiple cellular processes. We further identify critical aspects of TNKS:binding partner interaction that may provide novel targets for TNKS inhibition, potentially aiding in the realization of TNKS inhibition therapy in the treatment of human disease

Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo

Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus–based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association.

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved