Biology subjects

Joensuu, M.

Publications and source records attributed to Joensuu, M..

14 recordsLinked to original sources

bioRxiv2026

A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides

Peptide-based fusion inhibitors are promising pharmaceuticals in the fight against enveloped viruses relying on membrane fusion for host infection. However, peptide therapeutic applications have long been hindered by their poor stability in vivo. Here, we discovered that peptide inhibitors with the wildtype sequence of the heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein are efficiently cleaved by Transmembrane Protease, Serine 2 (TMPRSS2), a key protease involved in the SARS-CoV-2 virus-cell fusion pathway. We then identified the corresponding cleavage sites and designed three protease-resistant peptides using ranking based on deep mutational scanning and natural occurrence. The three candidates all exhibit inhibitory activity in a cell-cell fusion assay. A high-resolution cryo-EM structure of the top candidate, HR2-NHN, bound to its HR1 target reveals the molecular basis for its potent activity. The top candidate of the cell-based screening assay significantly improved efficacy relative to the wildtype peptide when administered 12 h before infection in both an authentic virus-cell infection assay and a mouse assay. More broadly, our results suggest that the design strategies for protease-resistant peptides could be applied to a broad spectrum of other enveloped viruses and pave the way for the development of safe, prophylactic antivirals that can be administered before exposure.

microbiology↗

bioRxiv2025

Omicron evolution drives increased ciliated cell tropism and dysfunction in nasal epithelia.

SARS-CoV-2 Omicron variants demonstrate greater replication efficiency in the upper respiratory tract than both ancestral virus and Delta variant, potentially due to stronger interactions with motile cilia in human nasal epithelial cells. Despite the emergence of numerous Omicron variants, there has been no comprehensive investigation comparing their interactions with ciliated cells. Here, we use differentiated primary human nasal epithelial cell (NEC) cultures to compare an ancestral isolate (QLD02) to Omicron variants BA.1, BA.5 and XBB. Transcriptomic analysis revealed that BA.5 and XBB infection, unlike BA.1 and QLD02, drives a distinct host response, defined by the profound downregulation of genes essential for cilium assembly and function at 48 hours post-infection. The BA.5/XBB signature was also characterized by the strong upregulation of pro-apoptotic and inflammatory pathways. Immunofluorescence of nucleocapsid-positive cells in NECs showed a significant 13-fold increase for BA.5 relative to QLD02. Additionally, BA.1 and BA.5 exhibited greater tropism for ciliated cells (7.6-fold for BA.1, 9-fold for BA.5) than QLD02. BA.5 and BA.1 infection were associated with elevated apoptosis in both infected and bystander cells, however, the total number of ciliated cells remained unchanged. Consistent with this, BA.5 infection significantly reduced DNAH5 outer dynein arm protein in the cilia, indicating dysfunction of cilia motility rather than overt cilia loss. Overall, we demonstrate a stepwise evolution at the nasal barrier: while enhanced ciliated cell tropism and elevated apoptosis are conserved features of Omicron infection, ciliary dysfunction, evident at both the transcriptional and protein level, is a more pronounced phenotype emerging with BA.5 and preserved in the XBB variant. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/689397v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@11b1b60org.highwire.dtl.DTLVardef@a25e0borg.highwire.dtl.DTLVardef@1cc3904org.highwire.dtl.DTLVardef@b35447_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Nanobinders for Synaptotagmin 1 enable the analysis of synapticvesicle dynamics in rodent and human models.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

neuroscience↗

bioRxiv2024

Saturated fatty acid-Coenzyme A supplementation restores neuronal energy levels and protein homeostasis in hereditary spastic paraplegia

Mitochondrial ATP production is fuelled by a fatty acid flux generated by phospholipase and triglyceride lipases in metabolically demanding tissues such as heart and liver, while the brain has long been believed to use almost solely glucose for energy. Phospholipase A1 enzyme DDHD2 is a major triglyceride lipase in the brain, and the loss of DDHD2 function results in a saturated free fatty acid (sFFA) imbalance and lipid droplet (LD) accumulation in the brain. The LD accumulation in neurons has been enigmatic as LDs are mainly considered to serve as a fuel storage. Here, we demonstrate that the loss of DDHD2 results in a mitochondrial respiratory dysfunction that leads to a significant decrease in ATP production and acetyl coenzyme A levels in neurons, even when the glycolytic breakdown of glycose occurs normally. Loss of DDHD2 also leads to a presynaptic defect as well as an imbalance in the global protein homeostasis in the neurons. These defects were rescued by external supplementation of the sFFA myristic acid coupled with its cofactor coenzyme A (Myr-CoA), indicating sFFA fuelling for neuronal {beta}-oxidation. We have thus discovered that the sFFAs released by the activity of DDHD2 play a central role in providing energy to fuel synaptic function. One Sentence SummaryFree fatty acids released by DDHD2 activity play a central role in maintaining neuronal energy levels and synaptic function.

neuroscience↗

bioRxiv2023

VAMP2/Munc18-1 domain 3a interaction controls the nanoscale reorganization underpinning vesicular priming

SNARE-mediated secretory vesicle (SV) exocytosis underpins neuronal communication. Munc18-1 orchestrates SNARE complex formation by controlling the opening of syntaxin-1A. How the SV-plasma membrane interface becomes fusion-competent at the nanoscale level is poorly understood. Here, we propose that the interaction of Munc18-1 with VAMP2 during vesicular docking triggers nanoscale re-organization which renders the SV-plasma membrane interface fusion-competent. We identified and mutated key residues in Munc18-1 domain 3a (A297 and T304) hypothesised to impair its interaction with VAMP2. Munc18-1A297H, and to a lesser extent Munc18-1T304H, constrained SVs on the plasma membrane and reduced stimulated secretion, under re-expression conditions in Munc18-1/2 double knockout neurosecretory cells. Moreover, the de-clustering of Munc18-1 in response to activity was lost for both mutants. The interaction of VAMP2 with the Munc18-1 domain 3a therefore controls the re-organization of the nanoscale environment of the docked SV-plasma membrane interface, fostering syntaxin-1A opening and Munc18-1 release to ensure that SNARE assembly only occurs within the confinement of docked vesicles.

neuroscience↗

bioRxiv2023

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.

microbiology↗

bioRxiv2023

Alternative cell entry mechanisms for SARS-CoV-2 and multiple animal viruses

The cell entry mechanism of SARS-CoV-2, the causative agent of the COVID-19 pandemic, is not fully understood. Most animal viruses hijack cellular endocytic pathways as an entry route into the cell. Here, we show that in cells that do not express serine proteases such as TMPRSS2, genetic depletion of all dynamin isoforms blocked the uptake and strongly reduced infection with SARS-CoV-2 and its variant Delta. However, increasing the viral loads partially and dose-dependently restored infection via a thus far uncharacterized entry mechanism. Ultrastructural analysis by electron microscopy showed that this dynamin-independent endocytic processes appeared as 150-200 nm non-coated invaginations and was efficiently used by numerous mammalian viruses, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, and rhinovirus. Both the dynamin-dependent and dynamin-independent infection of SARS-CoV-2 required a functional actin cytoskeleton. In contrast, the alphavirus Semliki Forest virus, which is smaller in diameter, required actin only for the dynamin-independent entry. The presence of TMPRSS2 protease rescued SARS-CoV-2 infection in the absence of dynamins. Collectively, these results indicate that some viruses such as canine parvovirus and SARS-CoV-2 mainly rely on dynamin for endocytosis-dependent infection, while other viruses can efficiently bypass this requirement harnessing an alternative infection entry route dependent on actin.

microbiology↗

bioRxiv2023

DDHD2 interacts with STXBP1 to mediate long-term memory via the generation of myristic acid

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brains lipid landscape remain largely unexplored. Saturated FFAs, particularly myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the DDHD2 isoform of PLA1 in mice reduced memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits, and markedly reduced saturated FFAs across the brain. DDHD2 was shown to bind to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of STXBP1 encephalopathy that is also associated with intellectual disability and motor dysfunction, we show that STXBP1 controls the targeting of DDHD2 to the plasma membrane and the generation of saturated FFAs in the brain. Our findings suggest key roles for DDHD2 and STXBP1 in the lipid metabolism underlying synaptic plasticity, learning and memory.

neuroscience↗

bioRxiv2023

The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, remains a global health concern despite vaccines, neutralizing antibodies, and antiviral drugs. Emerging mutations can reduce the effectiveness of these treatments, suggesting that targeting host cell factors may be a valuable alternative. N-myristoyltransferases (NMT) are essential enzymes for protein N-myristoylation, affecting stability, interaction, localization, and function of numerous proteins. We demonstrate that selective inhibition of host cell NMT decreases SARS-CoV-2 infection by 90% in human lung and primary nasal epithelial cells, and choroid plexus-cortical neuron organoids. NMT inhibition does not affect viral entry, replication or release, but impairs the maturation and incorporation of viral envelope proteins into newly assembled virions, leading to compromised infectivity of released virions. The inhibition of host NMT triggers a Golgi-bypassing pathway for SARS-CoV-2 progeny virion egress, which occurs through endoplasmic reticulum and lysosomal intermediates.

microbiology↗

bioRxiv2022

Surface SV2A-Syt1 nanoclusters act as a sequestration hub that limits dynamin-1 recruitment and targeting to recycling synaptic vesicles

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been postulated as a pre-assembly mechanism for endocytosis - ensuring high-fidelity retrieval. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through interaction of its C2B domain with SV2A, that are sensitive to mutations in this domain (Syt1K326A/K328A) and knocking down SV2A. SV2A co-cluster with Syt1 and blocking SV2As cognate interaction with Syt1 (SV2AT84A) also decreased SV2A clustering. Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced plasma membrane recruitment of key endocytic protein dynamin-1, leading to accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. SV2A-Syt1 surface nanoclusters therefore negatively regulate the rate of their own re-entry into recycling SVs by controlling the recruitment of the endocytic machinery.

neuroscience↗

bioRxiv2022

Synaptotagmin 1 mediates toxicity of botulinum neurotoxin type A

The extreme neurotoxicity of botulinum neurotoxins (BoNTs), broadly used as a therapeutics, is mediated by their direct binding to two plasma membrane receptors: polysialogangliosides (PSGs) and serotype-dependently either synaptotagmin 1/2 (Syt1/2) or synaptic vesicle glycoprotein 2 (SV2). Here, we demonstrate that although BoNT/A serotype binds directly to PSG and SV2, its neurotoxicity depends on Syt1. Using single-molecule superresolution microscopy of pathological concentrations of BoNT/A holotoxins, we demonstrate that the toxin binds to a preassembled PSG-Syt1 complex that forms nanoclusters with SV2 on the plasma membrane of hippocampal neurons. This coincidental interaction controls the selective endocytic targeting of BoNT/A into synaptic vesicles, leading to incapacitation of neuronal communication. Our results suggest that Syt1-SV2 plasma membrane nanoclusters may acts as a common target for various BoNT serotypes. One-Sentence SummaryBoNT/A hijacks an intrinsic tripartite PSG-Syt1-SV2 endocytic sorting mechanism to incapacitate neuronal communication

neuroscience↗

bioRxiv2021

SV2A-Syt1 interaction controls surface nanoclustering and access to recycling synaptic vesicles

Following exocytosis, the recapture of vesicular proteins stranded at the plasma membrane in recycling synaptic vesicles (SVs) is essential to sustain neurotransmission. Nanoclustering is emerging as a mechanism through which proteins may be pre-assembled prior to endocytosis, to ensure high fidelity of retrieval for subsequent rounds of vesicle fusion. Here, we used single molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A). Syt1 forms surface nanoclusters through interaction of its C2B domain (K326/K328) with SV2A, as demonstrated by mutating Syt1 (K326A/K328A) and knocking down endogenous SV2A. Blocking cognate interaction with Syt1 (SV2AT84A) also decreased SV2A clustering. Impaired nanoclustering of Syt1 and SV2A leads to accelerated endocytosis of Syt1, altered intracellular sorting and decreased trafficking of Syt1 to a Rab5-positive endocytic pathway. We conclude that the interaction between SV2A and Syt1 locks both molecules into surface nanoclusters, controlling their entry into recycling SVs.

neuroscience↗

bioRxiv2021

MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING

Single-molecule localization microscopy (SMLM) techniques are emerging as vital tools to unravel the nanoscale world of living cells. However, current analysis methods primarily focus on defining spatial nanoclusters based on detection density, but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in videogames to effectively detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to perform SMLM data analysis and determine whether the bounding boxes of individual molecular trajectories overlap, as a measure of their potential membership in nanoclusters. Extending the spatial indexing into the time dimension allows unique resolution of spatial nanoclusters into multiple spatiotemporal clusters. We have validated this approach using synthetic and SMLM-derived data. Quantitative characterization of recurring nanoclusters allowed us to demonstrate that both syntaxin1a and Munc18-1 molecules transiently cluster in hotspots on the neurosecretory plasma membrane, offering unprecedented insights into the dynamics of these protein which are essential to neuronal communication. This new analytical tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), has been implemented as a free and open-source Python graphic user interface.

biophysics↗

bioRxiv2020

Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system

The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Understanding how SARS-CoV-2 enters and spreads within human organs is crucial for developing strategies to prevent viral dissemination. For many viruses, tissue tropism is determined by the availability of virus receptors on the surface of host cells2. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a host receptor, yet, their tropisms differ3-5. Here, we found that the cellular receptor neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was inhibited by a monoclonal blocking antibody against the extracellular b1b2 domain of NRP1. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial cells and in the epithelial cells facing the nasal cavity. Neuropathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected NRP1-positive cells in the olfactory epithelium and bulb. In the olfactory bulb infection was detected particularly within NRP1-positive endothelial cells of small capillaries and medium-sized vessels. Studies in mice demonstrated, after intranasal application, NRP1-mediated transport of virus-sized particles into the central nervous system. Thus, NRP1 could explain the enhanced tropism and spreading of SARS-CoV-2.

microbiology↗