Biology subjects

Stromgaard, K.

Publications and source records attributed to Stromgaard, K..

9 recordsLinked to original sources

bioRxiv2026

Protein semi-synthesis enables real-time optical tracking of intracellular conformational changes during sodium channel inactivation

Dynamic conformational changes in intracellular domains fundamentally affect the function and pharmacology of many membrane proteins. For example, sodium influx through the cardiac voltage-gated sodium channel (NaV1.5) is rapidly terminated through conformational changes that result in pore closure, a transition known as inactivation. Inactivation involves NaV1.5 intracellular regions, particularly the DIII-DIV linker containing the IFM particle (Isoleucine-Phenylalanine-Methionine) and its dysfunction is a major cause of cardiac arrythmias. However, the conformational changes involved in inactivation and their modulation by auxiliary proteins and clinically used drugs remain incompletely characterized, partly because live-cell, site-specific labeling of intracellular regions with small fluorescent dyes remains challenging. Here, we combine live-cell protein semi-synthesis with voltage-clamp fluorometry (VCF), to track intracellular conformational dynamics of the cardiac sodium channel NaV1.5 and monitor their voltage dependence and kinetics in real time. We identify intracellular conformational changes involved in both fast and steady-state inactivation of NaV1.5 and show that both lidocaine and auxiliary proteins affect the kinetics of conformational changes of the DIII-DIV linker. Our work establishes the combination of protein semi-synthesis and voltage-clamp fluorometry as a powerful approach to dissect intracellular conformational changes in membrane proteins.

biophysics↗

bioRxiv2025

The SorCS2-derived macrocycle TT-P34 drives neuroprotection in animal models of neurodegeneration

Mitochondria are critical for sustaining the high energy demands of neuronal activity and their dysregulation is a hallmark of neurodegeneration. Targeting pathways of neurotrophic signaling is a well-established therapeutic strategy to enhance mitochondrial function and mitigate neurodegeneration. The VPS10p domain receptor, SorCS2, has recently emerged as a receptor with neurotrophic signaling capabilities. Here, we design and develop novel SorCS2-derived macrocyclic peptides mimicking receptor activation in vivo. We show that SorCS2-peptides enhance both neurotrophic support and boost metabolism by activating CREB and AMPK in a CAMKK2-dependent manner. This leads to upregulation of the key transcription factors PGC1 and TFEB and consequentially mitochondrial biogenesis. Furthermore, we show that the lipidated SorCS2 macrocycle, TT-P34, rescues motor behavioral deficits and preserves synaptic and mitochondrial signatures in the zQ175 mouse model of Huntingtons Disease. In addition, treating a MPTP-induced mouse model of Parkinsons Disease leads to amelioration of behavioral deficits and reduction of dopaminergic loss. Finally, we demonstrate that TT-P34 crosses the blood-brain barrier in non-human primates, and estimate human therapeutic dosing by pharmacodynamic modelling. Together, our findings support the use of TT-P34 as a novel disease-modifying therapy targeting SorCS2-receptor signaling to prevent neurodegeneration.

neuroscience↗

bioRxiv2023

DLG1 functions upstream of SDCCAG3 and IFT20 to control targeting of polycystin-2 to the primary cilium

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

cell biology↗

bioRxiv2022

Tight control of the APP-Mint1 interaction in regulating amyloid production

Generation of amyloid-beta (A{beta}) peptides through the proteolytic processing of the amyloid precursor protein (APP) is one pathogenic event in Alzheimers disease (AD). APP is a type I transmembrane protein and endocytosis of APP mediated by the endocytic YENPTY sequence is a key step in A{beta} generation. We and others have found that Mints, a family of cytosolic adaptor proteins, directly binds to the YENPTY motif of APP via phosphotyrosine binding (PTB) domain of Mints, facilitates APP trafficking and processing. We also show mutation of Tyr633 of Mint1 (Mint1Y633A) enhances APP binding and processing. Now, we created a low-affinity Mint1 mutant that targets two conserved residues, Tyr549 and Phe610 (Mint1Y549A/F610A), that reduced APP binding. Here, we investigate how perturbing the APP-Mint1 interaction alters APP and Mint1 cellular dynamics as well as Mint1s interaction with its other binding partners. We show that Mint1Y633A increased binding affinity specifically for APP and presenilin1, enhanced APP endocytosis, and A{beta} secretion in primary neurons. Conversely, Mint1Y549A/F610A exhibited reduced APP affinity and A{beta} secretion. In fact, the effect of Mint1Y549A/F610A on A{beta} release was greater compared to knocking down all three Mint proteins, supporting targeting APP-Mint1 interaction as a potential AD therapeutic.

neuroscience↗

bioRxiv2022

A syntenin inhibitor blocks endosomal entry of SARS-CoV-2 and a panel of RNA viruses

Viruses are dependent on interactions with host factors in order to efficiently establish an infection and replicate. Targeting such interactions provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking, and has previously been shown to be important for HPV infection. Here we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits SARS-CoV-2 infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection, and strongly reduced flavivirus infection, which are completely dependent on receptor mediated endocytosis for their entry. In conclusion, we have identified a novel pan-viral inhibitor that efficiently target a broad range of RNA viruses.

biochemistry↗

bioRxiv2022

Soluble amyloid-β precursor peptide does not regulate GABAB receptor activity

Amyloid-{beta} precursor protein (APP) regulates neuronal activity through the release of secreted APP (sAPP) acting at cell-surface receptors. APP and sAPP were reported to bind to the extracellular sushi domain 1 (SD1) of GABAB receptors (GBRs). A 17 amino-acid peptide (APP17) derived from APP was sufficient for SD1 binding and shown to mimic the inhibitory effect of sAPP on neurotransmitter release and neuronal activity. The functional effects of APP17 and sAPP were similar to those of the GBR agonist baclofen and blocked by a GBR antagonist. These experiments led to the proposal that sAPP activates GBRs to exert its neuronal effects. However, whether APP17 and sAPP indeed influence classical GBR signaling pathways in heterologous cells was not analyzed. Here, we confirm that APP17 binds to GBRs with nanomolar affinity. However, biochemical and electrophysiological assays indicate that APP17 does not influence GBR activity in heterologous cells. Moreover, we found no evidence for APP17 regulating K+ currents in cultured neurons, neurotransmitter release in brain slices, or neuronal activity in vivo. Our results show that APP17 is not a functional GBR ligand and indicate that sAPP exerts neuronal effects through receptors other than GBRs.

neuroscience↗

bioRxiv2021

Site-specific phosphorylation of PSD-95 dynamically regulates the post-synaptic density as observed by phase separation

Postsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes, and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD. Graphical Abstract: Phosphorylation of PSD-95. O_LIIllustration of the interaction between the PDZ domains of PSD-95 and the NMDA receptor (NMDAR) (PDB: 4PE5) at the plasma membrane. PSD-95 interacts with the AMPA receptor (AMPAR) (PDB: 3KG2) through Stargazin (PDB: 5KBU). Using the software Ranch, a model of FL PSD-95 was generated using known structural domains of PSD-95 connected via a fully-flexible linker (PDZ1-2, PDB: 3GSL; PDZ3, PDB: 5JXB; SH3-GK, PDB: 1KJW). C_LIO_LIIllustration of the effect of phosphorylation of PSD-95 on the formation of condensates at the PSD. PSD-95 can phase separate with receptors and intracellular proteins, and phosphorylation is can affect this interaction and lead to alterations, both negative and positive, in the formation of condensates. C_LI

biochemistry↗

bioRxiv2021

Bi-directional protein-protein interactions control liquid-liquid phase separation of PSD-95 and its interaction partners

The organization of the postsynaptic density (PSD), a protein-dense semi-membraneless organelle, is mediated by numerous specific protein-protein interactions (PPIs) which constitute a functional post-synapse. Postsynaptic density protein 95 (PSD-95) interacts with a manifold of proteins, including the C-terminal of transmembrane AMPA receptor (AMAPR) regulatory proteins (TARPs). Here, we uncover the minimal essential peptide responsible for the stargazin (TARP-{gamma}2) mediated liquid-liquid phase separation (LLPS) formation of PSD-95 and other key protein constituents of the PSD. Furthermore, we find that pharmacological inhibitors of PSD-95 can facilitate formation of LLPS. We found that in some cases LLPS formation is dependent on multivalent interactions while in other cases short peptides carrying a high charge are sufficient to promote LLPS in complex systems. This study offers a new perspective on PSD-95 interactions and their role in LLPS formation, while also considering the role of affinity over multivalency in LLPS systems.

biochemistry↗

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↗