Biology subjects

Colecraft, H. M.

Publications and source records attributed to Colecraft, H. M..

6 recordsLinked to original sources

bioRxiv2024

Decoding polyubiquitin regulation of KV7. 1 functional expression with engineered linkage-selective deubiquitinases

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the ubiquitin code that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP- targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.

molecular biology↗

bioRxiv2024

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins-- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis-- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite-- as revealed by a 3.1 [A] cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

biophysics↗

bioRxiv2023

A Genetically Encoded Actuator Selectively Boosts L-type Calcium Channels in Diverse Physiological Settings

L-type Ca2+ channels (CaV1.2/1.3) convey influx of calcium ions (Ca2+) that orchestrate a bevy of biological responses including muscle contraction and gene transcription. Deficits in CaV1 function play a vital role in cardiac and neurodevelopmental disorders. Yet conventional pharmacological approaches to upregulate CaV1 are limited, as excessive Ca2+ influx leads to cytotoxicity. Here, we develop a genetically encoded enhancer of CaV1.2/1.3 channels (GeeC) to manipulate Ca2+ entry in distinct physiological settings. Specifically, we functionalized a nanobody that targets the CaV macromolecular complex by attaching a minimal effector domain from a CaV enhancer--leucine rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC evoked a 3-fold increase in L-type current amplitude. In neurons, GeeC augmented excitation-transcription (E-T) coupling. In all, GeeC represents a powerful strategy to boost CaV1.2/1.3 function in distinct physiological settings and, in so doing, lays the groundwork to illuminate new insights on neuronal and cardiac physiology and disease.

physiology↗

bioRxiv2022

Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

The slow delayed rectifier potassium current, IKs, conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, IKs is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic upregulation of IKs requires recruitment of PKA holoenzyme (two regulatory- RI or RII- and two catalytic C subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9). Mutations in Q1 or AKAP9 that abolish their functional interaction result in long QT syndrome type 1 and 11, respectively, which increases the risk of sudden cardiac death during exercise. Here, we investigated the utility of a targeted protein phosphorylation (TPP) approach to reconstitute PKA regulation of IKs in the absence of AKAP9. Targeted recruitment of endogenous C to E1-YFP using a GFP/YFP nanobody (nano) fused to RII enabled acute cAMP-mediated enhancement of IKs, reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RII or C to Q1-YFP constitutively inhibited IKs by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed distinct phosphorylation sites are modified by C targeted to Q1-YFP compared to free C. Thus, functional outcomes of synthetically recruited PKA on IKs regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of IKs by phosphorylation across different spatial and time scales, and suggest a TPP approach to develop new drugs to prevent exercise-induced sudden cardiac death.

physiology↗

bioRxiv2021

Human stem cell model of HNF1A deficiency shows uncoupled insulin to C-peptide secretion with accumulation of abnormal insulin granules

Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3 (MODY3), the most prevalent form of monogenic diabetes. We generated stem cell-derived pancreatic endocrine cells from human embryonic stem cells (hESCs) with induced hypomorphic mutations in HNF1A. Using these cells, we show that HNF1A orchestrates a transcriptional program required for distinct aspects of {beta}-cell fate and function. During islet cell differentiation, HNF1A deficiency biases islet endocrine cells towards an -cell fate associated with PAX4 down-regulation. HNF1A- deficient {beta}-cells display impaired basal and glucose stimulated-insulin secretion in association with a reduction in CACNA1A and intracellular calcium levels, and impaired insulin granule exocytosis in association with SYT13 down-regulation. Knockout of PAX4, CACNA1A and SYT13 reproduce the relevant phenotypes. Reduction of insulin secretion is associated with accumulation of enlarged secretory granules, and altered stoichiometry of secreted insulin to C-peptide. In HNF1A deficient {beta}-cells, glibenclamide, a sulfonylurea drug used in the treatment of MODY3 patients, increases intracellular calcium to levels beyond those achieved by glucose, and restores C-peptide and insulin secretion to a normal stoichiometric ratio. To study HNF1A deficiency in the context of a human disease model, we also generated stem cell-derived pancreatic endocrine cells from two MODY3 patients induced pluripotent stem cells (iPSCs). While insulin secretion defects are constitutive in cells with complete HNF1A loss of function, {beta}-cells heterozygous for hypomorphic HNF1A mutations are initially normal, but lose the ability to secrete insulin and acquire abnormal stoichiometric secretion ratios. Importantly, the defects observed in these stem cell models are also seen in circulating proportions of insulin:C-peptide in nine MODY3 patients. One sentence of summaryDeficiency of the transcription factor HNF1A biases islet endocrine cell fate towards -cells, impairs intracellular calcium homeostasis and insulin exocytosis, alters the stoichiometry of insulin to C-peptide release, and leads to an accumulation of abnormal insulin secretory granules in {beta}-cells.

cell biology↗

bioRxiv2018

Targeting the CaV - interaction yields a selective antagonist of the N-type CaV2.2 channel with broad antinociceptive efficacy

Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of , {beta} and 2{delta} subunits. The {beta}-subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming subunit of CaV. We targeted the high-affinity protein-protein interface of CaV{beta}s pocket within the CaV-subunit. Structure-based virtual screening of 50,000 small molecule library docked to the {beta}-subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (compound 45). This small molecule bound to CaV{beta} and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion (DRG) sensory neurons, decreased pre-synaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory post-synaptic potentials (sEPSC), and inhibited release of the nociceptive neurotransmitter calcitonin gene related peptide (CGRP) from spinal cord. 45 was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (post-surgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. 45 did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. 45, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaV-{beta} function and ultimately be developed as a non-opioid therapeutic for chronic pain.

neuroscience↗