Publications by Year: 2018

Dendritic cell-expressed common gamma-chain recruits IL-15 for trans-presentation at the murine immunological synapse
Beilin, C., et al. Dendritic cell-expressed common gamma-chain recruits IL-15 for trans-presentation at the murine immunological synapse. Wellcome Open Res 3, 84 (2018).Abstract
Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses.  Here we examine the importance of γc for myeloid dendritic cell (DC) function. We utilize a combination of DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular associations or cytoskeletal reorganization following MHCII ligation. These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.
A tandem mass spectrometry sequence database search method for identification of O-fucosylated proteins by mass spectrometry
Swearingen, K.E., et al. A tandem mass spectrometry sequence database search method for identification of O-fucosylated proteins by mass spectrometry. J Proteome Res (2018).Abstract
Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to re-analyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.
Fusion surface structure, function, and dynamics of gamete fusogen HAP2
Feng, J., et al. Fusion surface structure, function, and dynamics of gamete fusogen HAP2. Elife 7, (2018).Abstract
HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.
A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System
Qin, Y., et al. A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System. Cell (2018).Abstract
Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β.
Ligand and cation-induced structural alterations of the leukocyte integrin LFA-1
Sen, M., Koksal, A.C., Yuki, K., Wang, J. & Springer, T.A. Ligand and cation-induced structural alterations of the leukocyte integrin LFA-1. J Biol Chem (2018).Abstract

In aI integrins including leukocyte function-associated antigen-1 (LFA-1), ligand-binding function is delegated to the aI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the aI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the aI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the aI domain is linked to headpiece opening and engenders a preferred aI domain orientation. Using small-angle Xray scattering (SAXS) and negative-stain EM we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+. Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized aI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Iglike domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1` helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1` helix movements are linked to ICAM-1 binding by the aI domain and to the extended-open conformation of the ectodomain. The LFA-1--ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton, and is consistent with the cytoskeletal force model of integrin activation.

Measuring integrin conformational change on the cell surface with super-resolution microscopy
Moore, T.I., Aaron, J., Chew, T.-L. & Springer, T.A. Measuring integrin conformational change on the cell surface with super-resolution microscopy. Cell Reports In Press, (2018). Publisher's VersionAbstract

We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.

High integrin αVβ6 affinity reached by hybrid domain deletion slows ligand-binding on-rate
Dong, X., et al. High integrin αVβ6 affinity reached by hybrid domain deletion slows ligand-binding on-rate. Proc Natl Acad Sci U S A (2018).Abstract
The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVβ6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVβ6 affinity, in contrast to β1 integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVβ6 on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αVβ6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVβ6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.
Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation
Le, V.Q., et al. Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation. EMBO J (2018).Abstract
Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.