Biology subjects

Johnson, S. M.

Publications and source records attributed to Johnson, S. M..

9 recordsLinked to original sources

bioRxiv2025

Fisheries trade and blue nutrient flows in Pacific Island Countries

Pacific Island Countries (PICs) are located in highly productive fishing regions that supply nutrient-rich fish to global markets. Marine fisheries are a critical source of protein and essential micronutrients for billions of people worldwide, supporting both local diets and global food security. International trade shapes modern blue food systems, influencing broader distributions and availability of these "blue nutrients." Yet, the structure of these trade networks, the nutritional composition of exported fisheries, and the implications for local food security remain poorly understood. Using global marine fisheries trade data from 1996-2020 combined with species-level nutrient compositions, we analyzed production, consumption, and nutrient balances for 12 PICs and used network analysis to characterize the structure of international blue nutrient flows. Here we show that many PICs experience persistent net losses of essential nutrients, particularly vitamin B12, protein, and fatty acids. This results in nutrient yields far below population needs, with only a few PICs (Vanuatu and Kiribati) meeting average requirements. Compared to global trade networks, the regional PICs trade is more modular, less reciprocal, and less clustered, implying reliance on a few intermediaries (Papua New Guinea and Fiji). Despite high domestic production and consumption, 54% of blue nutrients are exported from PICs rather than retained locally, leaving local nutritional deficits unaddressed even as PICs supply global seafood markets. Combining trade, nutrient, and network analyses can help inform strategies to increase nutrient retention, strengthen food security, and support resilience for PICs in the modern blue food economy.

ecology↗

bioRxiv2024

A Chaperonin Complex Regulates Organelle Proteostasis in Malaria Parasites

The apicoplast of Plasmodium parasites serves as a metabolic hub that synthesize essential biomolecules. Like other endosymbiotic organelles, 90% of the apicoplast proteome is encoded by the cell nucleus and transported to the organelle. Evidence suggests that the apicoplast has minimal control over the synthesis of its proteome and therefore it is unclear how organelle proteostasis is regulated. Here, we identified and investigated a large and conserved chaperonin (CPN) complex with a previously unknown function. Using genetic tools, we demonstrated that ablation of the apicoplast CPN60 subunit leads to parasite death due to organellar damage, immediately within its first replication cycle, deviating from the delayed death phenotype commonly observed for apicoplast translation inhibitors. Unlike its close orthologues in other prokaryotic and eukaryotic cells, CPN60 is not upregulated during heat shock (HS) and does not affect HS response in the parasite. Instead, we found that it is directly involved in proteostasis through interaction with the Clp (caseinolytic protease) proteolytic complex. We showed that CPN60 physically binds both the active and inactive forms of the Clp complex, and manipulates its stability. A computational structural model of a possible interaction between these two large complexes suggests a stable interface. Finally, we screened a panel of inhibitors for the bacterial CPN60 orthologue GroEL, to test the potential of chaperonin inhibition as antimalarial. These inhibitors demonstrated an anti-Plasmodium activity that was not restricted to apicoplast function, with additional targets outsides of this organelle. Taken together, this work reveals how balanced activities of proteolysis and refolding safeguard the apicoplast proteome, and is essential for organelle biogenesis. Author SummaryThe cell of the human malaria parasite Plasmodium falciparum has a unique organelle called the apicoplast that produces essential metabolites, but it is unclear how it maintains a stable proteome. Here, we address the question of organelle proteostasis by investigating the function of a large chaperonin complex and its main subunit CPN60. We show that CPN60 mutants die due to organellar damage immediately within the first replication cycle, avoiding the typical apicoplast-delayed cell death. We demonstrate that it binds and stabilizes another large proteolytic complex and use computational predicting tools to demonstrate how a stable interface is attained. We use bacterial inhibitors to explore their potential as an antimalarial drug target. This study reveals how balanced refolding and proteolysis safeguard the apicoplast proteome and opens a new avenue for antimalarial drug discovery.

microbiology↗

bioRxiv2023

Substrate-Specific Function of PNPLA3 Facilitates Hepatic VLDL-Triglyceride Secretion During Stimulated Lipogenesis

The I148M variant of PNPLA3 is strongly linked to hepatic steatosis. Evidence suggests a gain-of-function role for the I148M mutant as an ATGL inhibitor, leaving the physiological relevance of wild-type PNPLA3 undefined. Here we show that PNPLA3 selectively degrades triglycerides (TGs) enriched in polyunsaturated fatty acids (PUFAs) independently of ATGL in cultured cells and mice. Lipidomics and metabolite tracing analyses demonstrated that PNPLA3 mobilizes PUFAs from intracellular TGs for phospholipid desaturation, supporting hepatic secretion of TG-rich lipoproteins. Consequently, mice with liver-specific knockout or acute knockdown of PNPLA3 both exhibited aggravated liver steatosis and concomitant decreases in plasma VLDL-TG, phenotypes that manifest only under lipogenic conditions. I148M-knockin mice similarly displayed impaired hepatic TG secretion during lipogenic stimulation. Our results highlight a specific context whereby PNPLA3 facilitates the balance between hepatic TG storage and secretion and suggest the potential contributions of I148M variant loss-of-function to the development of hepatic steatosis in humans. Summary StatementWe define the physiological role of wild type PNPLA3 in maintaining hepatic VLDL-TG secretion.

physiology↗

bioRxiv2023

Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease I (APE1) acts as both an endonuclease and a redox factor to ensure cell survival. The two activities require different conformations of APE1. As an endonuclease, APE1 is fully folded. As a redox factor, APE1 must be partially unfolded to expose the buried residue Cys65, which reduces transcription factors including AP-1, NF-{kappa}B, and HIF-1 and thereby enables them to bind DNA. To determine a molecular basis for partial unfolding associated with APE1s redox activity, we characterized specific interactions of a known redox inhibitor APX3330 with APE1 through waterLOGSY and 1H-15N HSQC NMR approaches using ethanol and acetonitrile as co-solvents. We find that APX3330 binds to the endonuclease active site in both co-solvents and to a distant small pocket in acetonitrile. Prolonged exposure of APE1 with APX3330 in acetonitrile resulted in a time-dependent loss of 1H-15N HSQC chemical shifts ([~]35%), consistent with partial unfolding. Regions that are partially unfolded include adjacent N- and C-terminal beta strands within one of the two sheets comprising the core, which converge within the small binding pocket defined by the CSPs. Removal of APX3330 via dialysis resulted in a slow reappearance of the 1H-15N HSQC chemical shifts suggesting that the effect of APX3330 is reversible. APX3330 significantly decreases the melting temperature of APE1 but has no effect on endonuclease activity using a standard assay in either co-solvent. Our results provide insights on reversible partial unfolding of APE1 relevant for its redox function as well as the mechanism of redox inhibition by APX3330. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/547112v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13f9fe2org.highwire.dtl.DTLVardef@19cbcborg.highwire.dtl.DTLVardef@1927c7aorg.highwire.dtl.DTLVardef@2a3cc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Recurrent hypoxia in a rat model of sleep apnea during pregnancy leads to microglia-dependent respiratory deficits and persistent neuroinflammation in adult male offspring

Sleep apnea (SA) during pregnancy is detrimental to the health of the pregnancy and neonate, but little is known regarding long-lasting consequences of maternal SA during pregnancy on adult offspring. SA is characterized by repeated cessations in breathing during sleep, resulting in intermittent hypoxia (IH). We show that gestational IH (GIH) in rats reprograms the male fetal neuroimmune system toward enhanced inflammation in a region- and sex-specific manner, which persists into adulthood. Male GIH offspring also had deficits in the neural control of breathing, specifically in the ability to mount compensatory responses to central apnea, an effect that was rescued by a localized anti-inflammatory or microglial depletion. Female GIH offspring appeared unaffected. These results indicate that SA during pregnancy sex- and region-dependently skews offspring microglia toward a pro-inflammatory phenotype, which leads to long-lasting deficits in the capacity to elicit important forms of respiratory neuroplasticity in response to breathing instability. These studies contribute to the growing body of recent evidence indicating that SA during pregnancy may lead to sex-specific neurological deficits in offspring that persist into adulthood.

neuroscience↗

bioRxiv2021

Engineered protein-small molecule conjugates empower selective enzyme inhibition

SO_SCPLOWUMMARYC_SCPLOWSpecific, potent ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. A fibronectin protein library was yeast displayed with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enriched specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibited potent, specific inhibition of carbonic anhydrase isoforms with superior efficacy to acetazolamide or protein alone including an 80-fold specificity increase and 9-fold potency gain. PriSMs were engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveals platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

bioengineering↗

bioRxiv2020

Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

Extracellular vesicles (EVs) are heterogeneous in size (30nm-10{micro}m), content (lipid, RNA, DNA, protein) and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs and examine EV marker expression and diameter using imaging flow cytometry. MethodsWe describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and TRPS. Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. ResultsUW228-2 cells prolifically release EVs of up to 6 {micro}m. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. ConclusionsWe have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9 and LAMP1) on individual EVs within a widely heterogeneous population. Taken together data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.

cell biology↗

bioRxiv2019

A revised 1.6 Å structure of the GTPase domain of the Parkinson’s disease-associated protein LRRK2 provides insights into mechanisms

Leucine-rich repeat kinase 2 (LRRK2) is a large 286 kDa multi-domain protein whose mutation is a common cause of Parkinsons disease (PD). One of the common sites of familial PD-associated mutations occurs at residue Arg-1441 in the GTPase domain of LRRK2. Previously, we reported that the PD-associated mutation R1441H impairs the catalytic activity of the GTPase domain thereby traps it in a persistently \"on\" state. More recently, we reported that the GTPase domain of LRRK2 exists in a dynamic dimer-monomer equilibrium where GTP binding shifts it to the monomeric conformation while GDP binding shifts it back to the dimeric state. We also reported that all of the PD-associated mutations at Arg-1441, including R1441H, R1441C, and R1441G, impair the nucleotide-dependent dimer-monomer conformational dynamics of the GTPase domain. However, the mechanism of this nucleotide-dependent conformational dynamics and how it is impaired by the mutations at residue Arg-1441 remained unclear. Here, we report a 1.6 [A] crystal structure of the GTPase domain of LRRK2. Our structure has revealed a dynamic switch region that can be differentially regulated by GTP and GDP binding. This nucleotide-dependent regulation is impaired when residue Arg-1441 is substituted with the PD-associated mutations due to the loss of its exquisite interactions consisting of two hydrogen bonds and a {pi}-stacking interaction at the dimer interface.\n\nSignificance StatementMutations in LRRK2 are associated with familial Parkinsons disease, so understanding its mechanism of actions and how they are changed by the disease-associated mutations is important for developing therapeutic strategies. This paper describes an atomic structure of the G-domain of LRRK2 revealing that the conformational dynamics of the switch regions are potentially important for its normal function. It further shows that a disease-associated mutation could lock the G domain in a persistently active-like conformation, thus perturbing its normal function.

biochemistry↗

bioRxiv2017

Tonic Inhibition is Abolished in GABAA Receptor γ2R43Q Knock-in Mice with Absence Epilepsy and Febrile Seizures.

The {gamma}2R43Q GABAA receptor mutation confers absence epilepsy in humans, and {gamma}2R43Q knock-in mice (RQ) display absence seizures and generalized spike-and-wave discharges reminiscent of their human counterparts. Previous work on several rodent models led to the conclusion that elevated tonic inhibition in thalamic neurons is necessary and sufficient to produce typical absence epilepsy. In contrast, here we used patch-clamp electrophysiology in brain slices to show that RQ mice entirely lack tonic inhibition in principal cells of layer II/III somatosensory cortex and ventrobasal thalamus. Additionally, protein quantification and multielectrode electrophysiology show that the mutation interferes with trafficking of GABAA receptor subunits involved in generating tonic currents, leading to increased cortical firing and decreased thalamic bursting rates. Together with previous work, our results suggest that an optimum level of tonic inhibition is required for normal thalamocortical function, such that deviations in either direction away from this optimum enhance susceptibility to absence seizures.

neuroscience↗