Biology subjects

Lemieux, M. J.

Publications and source records attributed to Lemieux, M. J..

12 recordsLinked to original sources

bioRxiv2026

Supercharging the calcium pump: Identification of an activation hotspot on SERCA by cryo-EM.

The sarco-endoplasmic reticulum Ca{superscript 2}-ATPase (SERCA) is a ubiquitous P-type ATPase that restores cytosolic Ca2+ to the sarco-endoplasmic reticulum. SERCA is essential for cardiac Ca2+ cycling and cellular energy metabolism. Several small molecules enhance SERCA function and show promise in models of metabolic and cardiovascular diseases. However, the structural basis for SERCA activation has remained unknown, hindering mechanism-driven lead optimization. Here we present cryo-EM structures of SERCA bound to two chemically distinct activators: the quinoline derivative CDN1163 (2.6 [A] resolution) and a benzofuran derivative UM-52 (3.1 [A] resolution). Biochemical assays show that both compounds stimulate Ca2+-dependent ATPase activity of SERCA without altering the apparent Ca{superscript 2} affinity. The structures reveal a previously unrecognized "activation hotspot" in the transmembrane domain, a shallow groove formed by helices M3 and M4 and capped by M1. Despite low chemical similarity, both activators occupy the same pocket and share conserved interactions with Ser265, Trp272, and Phe296. These residues are unique to SERCA and help explain selectivity relative to other P-type ATPases. Activator binding stabilizes a catalytically competent conformation, shifting SERCA toward an E1-like state poised for ATP binding and coordinated movements of the M1-M4 bundle and the cytosolic domains. Notably, density consistent with a detergent acyl chain bridges an otherwise open cavity adjacent to the compound, suggesting that altered protein-lipid interactions may contribute to activation. Together, these findings define a structural framework for SERCA activation and provide a blueprint for rational design of next-generation SERCA activators. SIGNIFICANCE STATEMENTSERCA pumps Ca{superscript 2} into the sarco-endoplasmic reticulum, enabling muscle relaxation and shaping calcium signals across tissues. Small-molecule SERCA activators improve cardiac and metabolic phenotypes in animal models, but drug development has been limited by the absence of a defined binding site and activation mechanism. We determined cryo-EM structures of SERCA bound to two distinct activators, CDN1163 and UM-52. Both compounds occupy a groove formed by transmembrane segments M3-M4, anchored by a hydrogen bond to the SERCA-specific Ser265 and aromatic contacts near Trp272 and Phe296. An acyl chain bridges a gap in the binding pocket toward M1, suggesting that protein-lipid coupling may play a role in SERCA activation. These results directly enable structure-mechanism guided design of next-generation selective SERCA activators.

biophysics↗

bioRxiv2025

Mechanistic insight into the regulation of virulence factor type 1 pili in pathogenic E. coli by rhomboid protease GlpG

With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the E. coli rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the assembly of type 1 pili, a virulence factor required for the colonization of eukaryotic cells. Since pili are critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of type 1 pili and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates.

biochemistry↗

bioRxiv2024

The ER Thioredoxin-Related Transmembrane Protein TMX2 Controls Redox-Mediated Tethering of ER-Mitochondria Contacts (ERMCS)

Thioredoxin-related transmembrane proteins (TMX) of the endoplasmic reticulum (ER) have emerged as key regulators of ER membrane properties. Within the ER lumen, TMX proteins and other ER redox enzymes determine oxidative conditions, which control the formation of ER-mitochondria membrane contacts (ERMCS) and determine their function. ERMCS exhibit cytoplasmic redox nanodomains, derived from ER and mitochondrial reactive oxygen species (ROS), whose mechanistic regulation is uncharacterized. Our research has identified the ER protein TMX2, which uses its unique cytosolic thioredoxin domain to prevent cytosolic sulfenylation of mitochondrial outer membrane proteins such as TOM70 through a functional interaction with peroxiredoxin-1 (PRDX1). By doing so, TMX2 interferes with the TOM70 ERMCS tethering function and reduces mitochondrial Ca2+ flux and metabolism. Recently, TMX2 mutations have been identified to cause a neurodevelopmental disorder with microcephaly, cortical malformations, and spasticity (NEDMCMS). Using TMX2-mutated NEDMCMS patient cells, we demonstrate that compromising TMX2 through mutation reproduces mitochondrial defects. In a fly in vivo model, TMX2 knockdown manifests predominantly in glial cells. Our results therefore provide important mechanistic insight into NEDMCMS and mechanistically link TMX2-mediated control of ERMCS to brain development and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/589228v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@809587org.highwire.dtl.DTLVardef@1d8ef0corg.highwire.dtl.DTLVardef@135fdc6org.highwire.dtl.DTLVardef@1a7f7b6_HPS_FORMAT_FIGEXP M_FIG C_FIG The transmembrane thioredoxin-related TMX2 prevents TOM70 sulfenylation at ERMCS, thus maintaining normal mitochondria metabolism in wild-type cells. TMX2 knockout leads to TOM70 sulfenylation and tight ERMCS formation. This then increases ROS production, unbalances mitochondrial lipids, and relatively shifts OXPHOS electron supply to complex II.

cell biology↗

bioRxiv2023

SARS-CoV-2 Mpro protease variants of concern display altered viral and host target processing but retain potency towards antivirals

Main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOC) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and subsequent significant decrease in cytokine secretion, providing evidence for alterations in escape of host-antiviral mechanisms. Accordingly, mutations associated with the highly virulent Delta VOC resulted in significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

biochemistry↗

bioRxiv2021

A sensitive and specific genetically encodable biosensor for potassium ions

Potassium ions (K+) play a critical role as an essential electrolyte in all biological systems. Here we report the crystal structure-guided optimization and directed evolution of an improved genetically encoded fluorescent K+ biosensor, GINKO2. GINKO2 is highly sensitive and specific for K+ and enables in vivo detection of K+ dynamics in multiple species.

bioengineering↗

bioRxiv2021

SARS-COV-2 Recombinant Receptor-Binding-Domain (RBD) Induces Neutralising Antibodies Against Variant Strains of SARS-CoV-2 and SARS-CoV-1

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them are targeting the spike protein (or virion) in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested the utility of SARS-CoV-2 spike RBD as a vaccine antigen and explore the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results indicate RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralisation of variants B1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

microbiology↗

bioRxiv2021

A genetically encoded fluorescent biosensor for extracellular L-lactate

To enable investigations of the emerging roles of cell-to-cell shuttling of L-lactate, we have developed an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. We demonstrate that this biosensor, designated eLACCO1.1, enables minimally invasive cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

molecular biology↗

bioRxiv2021

N-Terminal finger stabilizes the reversible feline drug GC376 in SARS-CoV-2 Mpro

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drugs properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drugs P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

biophysics↗

bioRxiv2020

Dwarf open reading frame (DWORF) peptide is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.

The cardiac sarcoplasmic reticulum calcium pump, SERCA, sequesters calcium in the sarco-endoplasmic reticulum (SR/ER) and plays a critical role in the contraction-relaxation cycle of the heart. A well-known regulator of SERCA in cardiac muscle is phospholamban (PLN), which interacts with the pump and reduces its apparent calcium affinity. A newly discovered SERCA regulatory subunit in cardiac muscle, dwarf open reading frame (DWORF), has added a new level of SERCA regulation. In this report, we modeled the structure of DWORF and evaluated it using molecular dynamics simulations. DWORF structure was modeled as a discontinuous helix with an unwound region at Pro15. This model orients an N-terminal amphipathic helix along the membrane surface and leaves a relatively short C-terminal transmembrane helix. We determined the functional regulation of SERCA by DWORF using a membrane reconstitution system. Surprisingly, we observed that DWORF directly activated SERCA by increasing its turnover rate. Furthermore, in-cell imaging of calcium dynamics demonstrated that DWORF increased SERCA-dependent ER calcium load, calcium reuptake rate, and spontaneous calcium release. Together, these functional assays suggest opposing effects of DWORF and PLN on SERCA function. The results agree with fluorescence resonance energy transfer experiments, which revealed changes in the affinity of DWORF for SERCA at low versus high cytosolic calcium concentrations. We found that DWORF has a higher affinity for SERCA in the presence of calcium, while PLN had the opposite behavior, a higher affinity for SERCA in low calcium. We propose a new mechanism for DWORF regulation of cardiac calcium handling in which DWORF directly enhances SERCA turnover by stabilizing the conformations of SERCA that predominate during elevated cytosolic calcium.

biophysics↗

bioRxiv2020

Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min-1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated {beta}-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

biochemistry↗

bioRxiv2020

Sarcolambans are phospholamban- and sarcolipin-like regulators of the sarcoplasmic reticulum calcium pump SERCA

From insects to humans, calcium signaling is essential for life. An important part of this process is the sarco-endoplasmic reticulum calcium pump SERCA, which maintains low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, this is a tightly controlled system where SERCA interacts with tissuespecific regulatory subunits such as phospholamban in cardiac muscle and sarcolipin in skeletal muscle. With the recent discovery of the sarcolambans, the family of calcium pump regulatory subunits also appears to be ancient, spanning more than 550 million years of evolutionary divergence from insects to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and range from vaguely phospholamban-like to vaguely sarcolipin-like. Here, our goal was to investigate select sarcolamban peptides for their ability to regulate calcium pump activity. For a side-by-side comparison of diverse sarcolamban peptides, we tested them against mammalian skeletal muscle SERCA1a. This allowed us to determine if the sarcolamban peptides mimic phospholamban and sarcolipin in their regulatory activities. Four sarcolamban peptides were chosen from different invertebrate species. Of these, we were able to express and purify sarcolamban peptides from bumble bee, water flea, and tadpole shrimp. Sarcolamban peptides were co-reconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. While all peptides were super-inhibitors of SERCA, they exhibited either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations were used to reveal novel features of insect versus mammalian calcium pumps and the sarcolamban regulatory subunits.

biochemistry↗

bioRxiv2020

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

The COVID-19 pandemic, attributed to the SARS-CoV-2 coronavirus infection, resulted in millions infected worldwide and an immediate need for antiviral treatments. The main protease (Mpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide and subsequent viral replication. Feline infectious peritonitis, a fatal infection in cats caused by a coronavirus, was successfully treated previously with a dipeptide-based protease inhibitor. Here we show this drug, GC376, and its analog GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of the SARS-CoV and SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 in cell culture, with EC50 values near one micromolar and little to no toxicity. These protease inhibitors are soluble, non-toxic, and bind reversibly. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals (cats). The work here lays the framework for their use in human trials for the treatment of COVID-19.

biochemistry↗