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Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

ABSTRACT: Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photoinactivate targeted proteins of interest. First, we show that protease-activated receptor 2 (PAR2), a G-protein-coupled receptor and an important pathway that leads to pain sensitization, can be photoinactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched off in laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Second, we demonstrate that the photoinactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood-brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification.

SUBMITTER: Kang P

PROVIDER: S-EPMC7096286 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

Kang Peiyuan P Li Xiaoqing X Liu Yaning Y Shiers Stephanie I SI Xiong Hejian H Giannotta Monica M Dejana Elisabetta E Price Theodore John TJ Randrianalisoa Jaona J Nielsen Steven O SO Qin Zhenpeng Z

ACS nano 20191024 11

Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold ...[more]

PMID: 31613606

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Project description:BackgroundPharmacologic modulation has previously shown that transient receptor potential canonical (TRPC) channels play an important role in the pathogenesis of malignant hyperthermia. This study tested the hypothesis that genetically suppressing the function of TRPC6 can partially ameliorate muscle cation dyshomeostasis and the response to halothane in a mouse model relevant to malignant hyperthermia.MethodsThis study examined the effect of overexpressing a muscle-specific nonconducting dominant-negative TRPC6 channel in 20 RYR1-p.R163C and 20 wild-type mice and an equal number of nonexpressing controls, using calcium- and sodium-selective microelectrodes and Western blots.ResultsRYR1-p.R163C mouse muscles have chronically elevated intracellular calcium and sodium levels compared to wild-type muscles. Transgenic expression of the nonconducting TRPC6 channel reduced intracellular calcium from 331 ± 34 nM (mean ± SD) to 190 ± 27 nM (P < 0.0001) and sodium from 15 ± 1 mM to 11 ± 1 mM (P < 0.0001). Its expression lowered the increase in intracellular Ca2+ of the TRPC6-specific activator hyperforin in RYR1-p.R163C muscle fibers from 52% (348 ± 37 nM to 537 ± 70 nM) to 14% (185 ± 11 nM to 210 ± 44 nM). Western blot analysis of TRPC3 and TRPC6 expression showed the expected increase in TRPC6 caused by overexpression of its dominant-negative transgene and a compensatory increase in expression of TRPC3. Although expression of the muscle-specific dominant-negative TRPC6 was able to modulate the increase in intracellular calcium during halothane exposure and prolonged life (35 ± 5 min vs. 15 ± 3 min; P < 0.0001), a slow, steady increase in calcium began after 20 min of halothane exposure, which eventually led to death.ConclusionsThese data support previous findings that TRPC channels play an important role in causing the intracellular calcium and sodium dyshomeostasis associated with RYR1 variants that are pathogenic for malignant hyperthermia. However, they also show that modulating TRPC channels alone is not sufficient to prevent the lethal effect of exposure to volatile anesthetic malignant hyperthermia-triggering agents.Editor’s perspective

Project description:BackgroundIndividuals with diabetes exhibit a higher risk of cardiovascular disease and mortality compared to healthy individuals. Following a transient ischemic attack (TIA) the risk of stroke and death increase further. Physical activity engagement after a TIA is an effective way of secondary prevention. However, there's a lack of research on how individuals with diabetes modify physical activity levels and how these adjustments impact survival post-TIA. This study aimed to determine the extent to which individuals with diabetes alter their physical activity levels following a TIA and to assess the impact of these changes on mortality.MethodsThis was a nationwide longitudinal study, employing data from national registers in Sweden spanning from 01/01/2003 to 31/12/2019. Data were collected 2 years retro- and prospectively of TIA occurrence, in individuals with diabetes. Individuals were grouped based on decreasing, remaining, or increasing physical activity levels after the TIA. Cox proportional hazards models were fitted to evaluate the adjusted relationship between change in physical activity and all-cause, cardiovascular, and non-cardiovascular mortality.ResultsThe final study sample consisted of 4.219 individuals (mean age 72.9 years, 59.4% males). Among them, 35.8% decreased, 37.5% kept steady, and 26.8% increased their physical activity after the TIA. A subsequent stroke occurred in 6.7%, 6.4%, and 6.1% of individuals, while death occurred in 6.3%, 7.3%, and 3.7% of individuals, respectively. In adjusted analyses, participants who increased their physical activity had a 45% lower risk for all-cause mortality and a 68% lower risk for cardiovascular mortality, compared to those who decreased their physical activity.ConclusionsPositive change in physical activity following a ΤΙΑ was associated with a reduced risk of mortality. Increased engagement in physical activity should be promoted after TIA, thereby actively supporting individuals with diabetes in achieving improved health outcomes.

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Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

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Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

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