Biology subjects

Hamm, H. E.

Publications and source records attributed to Hamm, H. E..

8 recordsLinked to original sources

bioRxiv2026

Membrane-gated SNARE zippering focuses energy for fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive stagewise membrane fusion by zippering into membrane-bridging four-helix bundles. Yet the conformations underlying successive fusion stages and the coupling of folding energy to bilayer remodeling remain unclear. Using optical tweezers, we measured the intermediates, energetics, kinetics, and force dependence of individual synaptic SNARE complexes assembled in cis on single membranes and in trans between apposed membranes. Membrane-anchored cis-SNAREs assembled through N-terminal and cooperative C-terminal/linker-domain transitions, whereas their transmembrane domains showed little intrinsic dimerization. Syntaxin retained membrane-dependent helical continuity through its linker domain before zippering was complete. PIP2 strengthened but slowed late zippering. In trans, membrane repulsion arrested single trans-SNARE complexes in a half-zippered state. G{beta}{gamma} further clamped this intermediate and inhibited late zippering; G-GDP, but not G-GTP{gamma}S, relieved the clamp, revealing a nucleotide-dependent mechanism for GPCR-mediated inhibition of neurotransmitter release. Modeling suggests that cooperative late zippering, syntaxin linker helicity, and concerted action of multiple SNAREs focus folding energy released over a long distance onto short-range membrane apposition. Thus, mechanically gated SNARE zippering is regulated by membrane forces, lipids, and regulatory proteins.

cell biology↗

bioRxiv2026

Targeting Protease-activated Receptor 4 (PAR4) Protects Against Acute Kidney Injury (AKI) in Ischemia Reperfusion Injury

Acute kidney injury (AKI) is a serious and common clinical syndrome that currently has no effective treatment. Emerging evidence links coagulation pathways to kidney injury, particularly through coagulation proteases. Protease-activated receptors (PARs) are a family of G-protein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N termini, exposing a tethered ligand that initiates receptor signaling. PARs have been shown to play a major role in inflammation, vascular regulation, and tissue injury. PARs play key roles in inflammation, vascular regulation, and tissue injury. Previous work from the Hamm laboratory demonstrated that PAR4 contributes to AKI progression, as PAR4 knockout mice were protected in both unilateral ureteral obstruction and ischemia-reperfusion-based models of kidney disease. In this study, we investigated the potential of a PAR4 antagonist, VU6073819, at mitigating AKI progression in an ischemia-reperfusion injury (IRI) mouse model. PAR4 antagonism not only alleviated kidney injury and inflammatory response, but it significantly improved the survival. These findings identify PAR4 as a promising therapeutic target for AKI.

pharmacology and toxicology↗

bioRxiv2026

Architecture of the Gβγ-prefusion SNARE complex reveals the molecular mechanism of inhibition of vesicle fusion

Presynaptic inhibitory GPCR (Gi/o GPCR) signaling is an essential regulatory mechanism in vertebrate physiology. Near the presynaptic active zone, Gi/o GPCR activation releases G-protein {beta}{gamma} heterodimers (G{beta}{gamma}) which act to inhibit synaptic vesicle fusion through either modulation of Ca2+ entry via voltage-gated Ca2+ channels, or by direct interactions with the core exocytotic machinery comprised of the ternary SNARE complex downstream of Ca2+ influx. The precise molecular mechanism underlying G{beta}{gamma}-SNARE mediated inhibition has remained unclear due to lack of structural data for the G{beta}{gamma}-SNARE complex. We address this long-standing question here by stabilizing the interaction between G{beta}1{gamma}2 and a pre-fusion ternary SNARE mimetic and determining the structure using single-particle cryo-EM. We used our cryo-EM envelope to build an atomic level prediction of the interaction interface. We validated key interaction sites predicted by our model at the C-terminus of SNAP-25 using site directed mutagenesis and biochemical affinity measurements. Additionally, we found that G{beta}1{gamma}2 and a fragment of the regulatory protein complexin can engage the pre-fusion SNARE complex simultaneously. On the basis of these results, we propose a model in which G{beta}1{gamma}2 acts on the partially zipped SNARE complex at a late stage in the vesicle docking and priming cycle. In the model, the amino-terminal coiled-coil of G{beta}1{gamma}2 forms an interface with the C-terminus of the target membrane SNARE (t-SNARE) complex to prevent complete incorporation of the vesicle SNARE (v-SNARE) into the core SNARE helical bundle, thus blocking vesicle approach to the plasma membrane. The {beta}-propeller domain of G{beta}1 may also sterically hinder vesicle approach. Together these results provide crucial structural insights into the mechanism of binding of G{beta}{gamma} to the SNARE complex, and lends essential insights into the critical role of GPCR signaling to the SNARE complex in modulating synaptic vesicle fusion.

biophysics↗

bioRxiv2022

G protein βγ subunits bind to and inhibit the function of multiple Qa- and Qb,c-SNARE isoforms.

While the ability of G protein {beta}{gamma} subunits (G{beta}{gamma}) to bind to and functionally inhibit the neuronal SNARE proteins Stx1A, SNAP25, and synaptobrevin in the presence of the calcium sensor synaptotagmin I is well documented, these three SNARE proteins, which form the core SNARE complex for synchronous evoked release in neurons, are but a subset of the larger family of SNARE proteins, which participate in many other exocytic processes within the cell and in other populations of secretory cells throughout the body, from which the release of neurotransmitters, hormones, and other factors is regulated by Gi/o-coupled GPCRs. The ability of G{beta}{gamma} to regulate these processes is unknown. To investigate the feasibility of this mechanism to inhibit SNARE function more broadly, we utilized a series of biochemical assays of binding and function with four Qa-SNAREs (Stx1A, Stx2, Stx3, and Stx4) and four Qb,c-SNAREs (SNAP25, SNAP23, SNAP29, and SNAP47) in tandem with the R-SNARE synaptobrevin, synaptotagmin I, and G{beta}{gamma}. G{beta}{gamma} was found to bind to multiple Qa-SNARE isoforms as well as SNAP23, and inhibit the lipid mixing function of these SNAREs, as well as SNAP29. Together, this data suggests a more broad role for the G{beta}{gamma}-SNARE pathway in the regulation of exocytosis beyond cells that express Stx1A or SNAP25.

biochemistry↗

bioRxiv2020

Specificity of Gβ and γ subunits to SNARE complex both at rest and after α2aadrenergic receptor stimulation

Though much is known about the various physiological functions of each GPCR and the specificity of G subunits, the specificity of G{beta}{gamma} activated by a given GPCR and activating each effector in vivo is not known. Previously, we identified different G{beta} and G{gamma} subunits interacting specifically with 2a-adrenergic receptors (2aAR). In this study, we examined its in vivo specificity to the soluble NSF attachment proteins (SNARE) complex in adrenergic (auto-2aAR) and non-adrenergic (hetero-2aAR) neurons. We applied a quantitative targeted multiple reaction monitoring proteomic analysis of G{beta} and G{gamma} subunits bound to the SNARE complex, and found only a subset of G{beta} and G{gamma} bound. Without stimulation of auto-2aAR, G{beta}1 and G{gamma}3 interacted with the SNARE complex. When auto-2aAR were activated, G{beta}1, G{beta}2, and G{gamma}3 were found. Further understanding of in vivo G{beta}{gamma} specificity to its effectors provides new insights into the multiplicity of genes for G{beta} and G{gamma}. SummarySpecific G{beta}{gamma} dimers interact with the SNARE complex following presynaptic 2aAR activation in both adrenergic and non-adrenergic neurons.

biochemistry↗

bioRxiv2020

Impairment of the Gβγ-SNAP25 brake on exocytosis enhances insulin action, protects against diet-induced obesity, and promotes adipocyte browning

Negative regulation of exocytosis from secretory cells throughout the body is accomplished through inhibitory signals from Gi/o G protein-coupled receptors by G{beta}{gamma} subunit inhibition of two common mechanisms: (i) decreased calcium entry and (ii) direct interaction of G{beta}{gamma} with the Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. We have previously shown that disabling the second mechanism with a truncation of SNAP25 (SNAP25{Delta}3/{Delta}3) decreases the affinity of G{beta}{gamma} for the SNARE complex, leaving exocytotic fusion as well as modulation of calcium entry intact but disabling GPCR inhibition of exocytosis. Here we report significant beneficial metabolic remodeling in mice carrying this mutation. Chow-fed SNAP25{Delta}3/{Delta}3 mice exhibit enhanced insulin sensitivity and increased beiging of white fat. In response to a high fat diet, the metabolic protection was amplified in SNAP25{Delta}3/{Delta}3 mice. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. This metabolic protection in SNAP25{Delta}3/{Delta}3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neurons exocytosis from SNAP25{Delta}3/{Delta}3 inguinal adipose depot resulted in enhanced and prolonged norepinephrine release. Thus, the G{beta}{gamma}-SNARE interaction represents a cellular mechanism that deserves further exploration as a new avenue for combatting metabolic disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/069138v4_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@2c1fa0org.highwire.dtl.DTLVardef@d076ceorg.highwire.dtl.DTLVardef@27e351org.highwire.dtl.DTLVardef@bce219_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2020

Critical Residues of Gβγ for the interaction with the SNARE Complex

The mechanisms and regulation of neurotransmitter release is a complex process involving many co-factors and proteins. One critical interaction is the regulation of exocytosis when G-protein {beta}{gamma} (G{beta}{gamma}) dimers bind to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complex. The complex is comprised of N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Herein we probe across the entire family of human G{beta} and G{gamma} proteins for residues critical for the interaction with SNARE, by systematically screening peptide sequences for their ability to bind to tSNARE. The coiled-coil region of G{beta}{gamma} showed high affinity to tSNARE, along with the propeller region of G{beta} on the opposite side from the coiled-coil region. Peptides based on G{beta}1{gamma}2, shown to have high affinity to SNARE, tSNARE were screened further by alanine scanning to probe for residues critical for binding to tSNARE. Full length G{beta}1{gamma}2 and SNARE were docked computationally using Rosetta, to examine the experimentally determined binding sites. Docking converged on two possible sites of interaction using two distinct regions of both G{beta}1{gamma}2 and SNARE.

biochemistry↗

bioRxiv2018

Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes.

Gi/o-coupled G-protein coupled receptors modulate neurotransmission presynaptically through inhibition of exocytosis. Release of G{beta}{gamma} subunits decreases the activity of voltage-gated calcium channels (VGCC), decreasing excitability. A less understood G{beta}{gamma}-mediated mechanism downstream of calcium entry is the binding of G{beta}{gamma} to SNARE complexes. Here, we create a mouse partially deficient in this interaction. SNAP25{Delta}3 homozygote animals are developmentally normalbut impaired gait and supraspinal nociception. They also have elevated stress-induced hyperthermia and impaired inhibitory postsynaptic responses to 2A-AR, but normal inhibitory postsynaptic responses to Gi/o-coupled GABAB receptor activation. SNAP25{Delta}3 homozygotes have deficits in inhibition of hippocampal postsynaptic responses to 5 HT1b agonists that affect hippocampal learning. These data suggest that Gi/o-coupled GPCR inhibition of exocytosis through the G{beta}{gamma}-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

neuroscience↗