12 research outputs found
DogCatcher allows loop-friendly protein-protein ligation
There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures
Global PIEZO1 Gain-of-Function Mutation Causes Cardiac Hypertrophy and Fibrosis in Mice
PIEZO1 is a subunit of mechanically-activated, nonselective cation channels. Gain-of-function PIEZO1 mutations are associated with dehydrated hereditary stomatocytosis (DHS), a type of anaemia, due to abnormal red blood cell function. Here, we hypothesised additional effects on the heart. Consistent with this hypothesis, mice engineered to contain the M2241R mutation in PIEZO1 to mimic a DHS mutation had increased cardiac mass and interventricular septum thickness at 8–12 weeks of age, without altered cardiac contractility. Myocyte size was greater and there was increased expression of genes associated with cardiac hypertrophy (Anp, Acta1 and β-MHC). There was also cardiac fibrosis, increased expression of Col3a1 (a gene associated with fibrosis) and increased responses of isolated cardiac fibroblasts to PIEZO1 agonism. The data suggest detrimental effects of excess PIEZO1 activity on the heart, mediated in part by amplified PIEZO1 function in cardiac fibroblasts
Force Sensing by Piezo Channels in Cardiovascular Health and Disease
Mechanical forces are fundamental in cardiovascular biology, and deciphering the mechanisms by which they act remains a testing frontier in cardiovascular research. Here, we raise awareness of 2 recently discovered proteins, Piezo1 and Piezo2, which assemble as transmembrane triskelions to combine exquisite force sensing with regulated calcium influx. There is emerging evidence for their importance in endothelial shear stress sensing and secretion, NO generation, vascular tone, angiogenesis, atherosclerosis, vascular permeability and remodeling, blood pressure regulation, insulin sensitivity, exercise performance, and baroreceptor reflex, and there are early suggestions of relevance to cardiac fibroblasts and myocytes. Human genetic analysis points to significance in lymphatic disease, anemia, varicose veins, and potentially heart failure, hypertension, aneurysms, and stroke. These channels appear to be versatile force sensors, used creatively to inform various force-sensing situations. We discuss emergent concepts and controversies and suggest that the potential for new important understanding is substantial
Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity
Piezo1 forms mechanically-activated non-selective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial apoptosis, with no effect on thrombospondin-1 (TSP1), a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without effect on Tsp1. In the endothelial cells, Piezo1 was required for normal expression of endothelial nitric oxide synthase (eNOS). The data suggest an endothelial-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability
Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress
KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice
Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM
Using FRET to Determine How Myo10 Responds to Force in Filopodia
Myosin 10 (Myo10) is an actin-based molecular motor that is essential for filopodia formation and likely senses tension through interactions with integrins in filopodial tips. It possesses a single α-helical (SAH) domain at the end of its canonical lever, which amplifies the movement of the motor. We have shown the SAH domain can contribute to lever function and possesses the properties of a constant force spring. Here we investigate whether the SAH domain plays a role in tension sensing and whether it becomes extended under load at the filopodial tip. Previously, we found that removing the entire SAH domain and short anti-parallel coiled coil (CC) region at the C-terminal end of the SAH does not prevent Myo10 from moving to filopodial tips in cells. Exploiting this, we generated recombinant forms of Myo10, in which a tension-sensing module (TSMod), comprising a FRET-pair YPet and mCherry separated by a linker sequence of amino acids was then inserted between the Myo10 motor and tail domains, so as to replace the SAH domain and CC region. The linker sequence comprised either a portion of the native SAH domain, or control sequences that were either short (x1: stiff) or long (x5: flexible) repeats of “GPGGA”. As additional controls we also placed the TSMod construct at the N-terminus, where it should not experience force. Our FRET measurements indicate that the SAH domain of Myo10 may become extended at when the protein is stalled at the filopodial tips, so the SAH domain may therefore act as a force sensor
DogCatcher allows loop-friendly protein-protein ligation
There are many efficient ways to connect proteins at termini. However, connecting at a loop is
difficult because of lower flexibility and variable environment. Here we have developed
DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide.
DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin.
We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction
rate 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein
terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However,
inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than
SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed
termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific
covalent labeling on cells in 1 minute. DogTag/DogCatcher reacts under diverse conditions, at
nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the
toolbox for creating protein architectures
Potent, selective, and subunit‐dependent activation of TRPC5 channels by a xanthine derivative
Background and purpose
TRPC1, TRPC4 and TRPC5 can form homo‐ or heterotetrameric, calcium‐permeable cation channels that have been implicated in various disease states. Recent research has yielded specific and potent xanthine‐based TRPC1/4/5 inhibitors. Here we investigated the possibility of xanthine‐based activation.
Experimental approach
An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesised and its activity was investigated using HEK cells over‐expressing TRPC4, TRPC5, TRPC4–C1, TRPC5–C1, TRPC1:C4 or TRPC1:C5, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 ion channel activities were measured by [Ca²⁺]i measurements and by patch‐clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4 or TRPM2.
Key results
AM237 potently activated TRPC5:C5 channels in [Ca²⁺]i measurements (EC₅₀ 15‐20 nM), and potentiated TRPC5:C5 activation by sphingosine‐1‐phosphate, yet suppressed TRPC5:C5 channel activity evoked by (‐)‐englerin A (EA). In patch‐clamp studies AM237 also activated TRPC5:C5 channels, with greater effect at positive voltages, but it was a weaker activator than EA. Pico145 competitively inhibited AM237‐induced TRPC5:C5 activation. In contrast, AM237 did not activate TRPC4:C4, TRPC4–C1, TRPC5–C1, TRPC1:C5 and TRPC1:C4 channels, nor native TRPC1:C4 channels in A498 cells, but potently inhibited EA‐dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4 or TRPM2 channels.
Conclusions and implications
This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine‐based small molecules can activate, potentiate or inhibit these channels depending on subunit composition
Kv1.3 voltage-gated potassium channels link cellular respiration to proliferation through a non-conducting mechanism
Cellular energy metabolism is fundamental for all biological functions. Cellular proliferation requires extensive metabolic reprogramming and has a high energy demand. The Kv1.3 voltage-gated potassium channel drives cellular proliferation. Kv1.3 channels localise to mitochondria. Using high-resolution respirometry, we show Kv1.3 channels increase oxidative phosphorylation, independently of redox balance, mitochondrial membrane potential or calcium signalling. Kv1.3-induced respiration increased reactive oxygen species production. Reducing reactive oxygen concentrations inhibited Kv1.3-induced proliferation. Selective Kv1.3 mutation identified that channel-induced respiration required an intact voltage sensor and C-terminal ERK1/2 phosphorylation site, but is channel pore independent. We show Kv1.3 channels regulate respiration through a non-conducting mechanism to generate reactive oxygen species which drive proliferation. This study identifies a Kv1.3-mediated mechanism underlying the metabolic regulation of proliferation, which may provide a therapeutic target for diseases characterised by dysfunctional proliferation and cell growth