Biology subjects

Andersen, J.

Publications and source records attributed to Andersen, J..

22 records · Page 2Linked to original sources

bioRxiv2020

High-throughput characterization of 309 photocrosslinker-bearing ASIC1a variants maps residues critical for channel function and pharmacology

Incorporation of non-canonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch-clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin-1 interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

biophysics↗

bioRxiv2020

The mitochondrial permeability transition pore activates a maladaptive mitochondrial unfolded protein response

Mitochondrial activity determines aging rate and the onset of chronic diseases. The mitochondrial permeability transition pore (mPTP) is a pathological pore in the inner mitochondrial membrane thought to be composed of the F-ATP synthase (complex V). OSCP, a subunit of F-ATP synthase, helps protect against mPTP formation. How the destabilization of OSCP may contribute to aging, however, is unclear. We have found that loss OSCP in the nematode Caenorhabditis elegans initiates the mPTP and shortens lifespan specifically during adulthood, in part via initiation of the mitochondrial unfolded protein response (UPRmt). Pharmacological or genetic inhibition of the mPTP inhibits the UPRmt and restores normal lifespan. Loss of the putative pore-forming component of F-ATP synthase extends adult lifespan, suggesting that the mPTP normally promotes aging. Our findings reveal how an mPTP/UPRmt nexus may contribute to aging and age-related diseases and how inhibition of the UPRmt may be protective under certain conditions.

cell biology↗

bioRxiv2019

Mechanism and site of action of big dynorphin on ASIC1a

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics. Significance StatementNeuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

biophysics↗

bioRxiv2019

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

cell biology↗