L’objectif de ma thèse était d’identifier, comme agents antinociceptifs potentiels, des toxines de venins originales par leur séquence et/ou leur provenance. Dans cette optique, un criblage à haut débit de deux banques de venins a été réalisé par des méthodes électrophysiologiques de "patch-clamp" automatique, sur des lignées cellulaires exprimant le sous-type neuronal hNaV1.7 de canaux sodium (versus celles exprimant le sous-type cardiaque hNaV1.5), une cible antidouleur validée génétiquement et fortement exprimée au niveau des neurones sensoriels primaires des ganglions de la racine dorsale, premier support de la transmission du message nociceptif.Le criblage de la première banque de venins (appartenant à Smartox Biotechnology) a permis l’identification et la caractérisation, par des approches structurales et fonctionnelles multi-échelles (de la cellule in vitro à l’organisme in vivo), de 2 peptides de venins d’araignées ayant des propriétés potentiellement antinociceptives : (1) la cyriotoxine-1a du venin de Cyriopagopus schioedtei, dont les propriétés fonctionnelles sont proches de celles des peptides appartenant à la famille 1 des toxines d’araignées inhibant les canaux sodium, et (2) la poecitoxine-1a du venin de Poecilotheria subfusca, qui présente une meilleure affinité pour le sous-type hCaV1.2 de canaux calcium que pour le sous-type hNaV1.7. Nous avons également mené une étude de "structure-activité" afin d’améliorer le profil de sélectivité de la phlotoxine-1 d’une araignée Phlogiellus, connue pour son activité antinocieptive. Finalement, nous avons mis en évidence une interaction directe entre l’huwentoxine-IV, déjà connue comme agent antinociceptif potentiel, et le sous-type neuronal NaV1.6, responsable d’effets neuromusculaires indésirables. Le criblage de la deuxième banque de venins (appartenant à SANOFI) a permis d’identifier des hits intéressants provenant de venins d’araignées et de scorpions non étudiés jusqu’à présent et ayant une séquence originale présentant peu d’homologie avec les séquences déjà connues. / The aim of my thesis was to identify original venom toxins, by their sequence and/or origin, as potential antinociceptive agents. In this context, a high-throughput screening of two venom libraries was performed, by automated patch-clamp electrophysiology, on cell lines expressing the hNaV1.7 neuronal subtype of sodium channels (versus those expressing the hNaV1.5 cardiac subtype), a genetically-validated and strongly expressed pain target in the primary sensory neurons of dorsal root ganglia, the first support of nociceptive message transmission.The screening of the first venom library (belonging to Smartox Biotechnology) allowed the identification and characterization, by structural and multiscale functional approaches (from the cell in vitro to the organism in vivo), of 2 peptides from spider venoms having potential antinociceptive properties : (1) cyriotoxin-1a from Cyriopagopus schioedtei venom, whose functional properties are close to those of peptides belonging to family 1 of spider toxins inhibiting sodium channels, and (2) poecitoxin-1a from Poecilotheria subfusca venom, which has a better affinity for the hCaV1.2 subtype of calcium channels than for the hNaV1.7 subtype. We also conducted a "structure-activity" study to improve the selectivity profile of phlotoxin-1 from a Phlogiellus spider, known for its antinociceptive activity. Finally, we evidenced a direct interaction between huwentoxin-IV, already known as a potential antinociceptive agent, and the NaV1.6 neuronal subtype which is the main cause of undesirable neuromuscular effects. The screening of the second venom library (belonging to SANOFI) allowed to identify interesting hits from spider and scorpion venoms, not studied until now, having an original sequence with little homology with already known sequences.

Les Ranunculales sont un ordre d’angiospermes d’environ 4500 espèces, incluant des plantes communes comme les boutons d’or et les coquelicots. Leurs fleurs sont très diversifiées et ont piqué l’intérêt des botanistes depuis des décennies. Les sept familles de l’ordre sont faciles à reconnaître sur le terrain, mais paradoxalement, certaines familles n’ont pas de synapomorphies et ont des états ancestraux peu clairs, comme les Ranunculaceae (~ 2500 espèces). Cette diversité florale peut être liée à la pollinisation et des innovations ont pu être guidées par cette interaction. De plus, les plantes de ce groupe produisent une grande variété de composés secondaires, certains dont les propriétés sont connues depuis fort longtemps (comme la morphine et le curare). Ces substances pourraient jouer un rôle dans la défense contre les herbivores. Les caractères floraux et chimiques sont donc de bons candidats pour produire des hypothèses adaptatives. Ici nous utilisons des méthodes analytiques modernes pour comprendre les patrons d’évolution expliquant la distribution actuelle de la diversité et l’évolution des fleurs et des toxines de Ranunculales. Un nouveau cadre phylogénétique synthétique a été produit avec des séquences publiées de 144 espèces. Seize caractères floraux et la présence de certains composés métaboliques ont été reconstruits sur cet arbre avec la méthode de parcimonie et la méthode de « Reversible Jump Monte Carlo Markov Chains ». Un nouveau scénario d’évolution florale a été produit, où les Ranunculales avaient une fleur ancestrale trimère avec trois cycles de tépales et où les ancêtres des familles ont évolué en perdant ou en différenciant des cycles du périanthe. Les patrons d’évolution des toxines montrent de la variabilité, certaines apparaissant et disparaissant aléatoirement (comme les saponines), d’autres étant des synapomorphies bien soutenues de certains clades (comme les diterpènes de type aconitine pour la tribu des Delphinieae). Ces résultats offrent une meilleure compréhension de l’histoire naturelle des Ranunculales. En outre, cet ordre est le groupe-frère de toutes les autres eudicotylédones, et a donc une position clef pour comprendre l’évolution précoce de ce clade. Ces résultats vont fournir une compréhension plus profonde des changements floraux et phytochimiques qui ont eu lieu à la base des eudicotylédones, qui contiennent 70% des angiospermes actuelles. / Ranunculales are an order of angiosperms comprising ca. 4,500 species including common plants like buttercups and poppies. Their flowers are highly diversified and have raised the interest of botanists for decades. Each of the seven families of the order is easily recognizable in the field, but paradoxically some families lack floral synapomorphies and have unclear ancestral states, like Ranunculaceae (ca. 2,500 species). This floral diversity may be linked to pollination and innovations may have been driven by this interaction. In addition, the plants of this group produce a variety of secondary compounds, some of which having been known for ages for their properties, like morphine or curare. Those substances could play a role in defense against herbivores. Both floral and chemical characters are thus good candidates to produce adaptive hypotheses. Here we used modern analytical methods to understand the the evolutionary patterns accounting for the extant distribution of diversity and evolution of Ranunculales flowers and toxins. A new synthetic phylogenetic framework of the Ranunculales based on already available sequences of 144 species was produced. Sixteen characters of the flower, and the presence of selected secondary metabolites were reconstructed on this tree using parsimony, and Reversible Jump Monte Carlo Markov Chains. A new scenario for floral evolution was produced, where Ranunculales had a trimerous perianth with three whorls of tepals ancestor, and the ancestors of the families evolved by losing and/or differentiating perianth whorls. The evolutionary patterns of toxins showed variability, some appearing and disappearing randomly (such as saponins), others being well-supported synapomorphies (such as aconitine-like diterpens for the tribe Delphinieae). These results offer a better understanding of the natural history of Ranunculales. Furthermore, this order is the sister-group of all other eudicots, and thus has a key position to understand the early evolution of this clade. These results will provide a deeper understanding of the floral and chemical changes that took place at the base of eudicots, which contains 70% of living angiosperms.

Enterohemorrhagic E. coli (EHEC) infections are a leading cause of foodborne illness in the United States. Shiga-like toxins are produced that can cause hemorrhagic colitis and can lead to dangerous complications, such as acute kidney injury and hemolytic uremic syndrome (HUS). There are currently no specific treatments for HUS, and therefore more research into EHEC and HUS needs to be done. Our study focuses on Shiga-like toxin induction of endoplasmic reticulum (ER) stress in in vitro and in vivo systems, using human monocyte-like THP-1 cells and a non-human primate model of HUS. We used qPCR to determine the levels of ER stress marker expression induced by both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) challenges. We also looked at ER stress marker expression in non-human primates that survived a lethal Stx2 challenge after being given a Stx2 binding tetravalent peptide. We expected to see increased ER stress marker expression in THP-1 cells challenged with both Shiga-like toxins and in animals that received lethal doses of the toxins. Although results were inconclusive for THP-1 cell experiments, our preliminary non-human primate data suggest that the timing of ER stress marker production is important, and Shiga-like toxins may suppress the unfolded protein response (UPR) in some baboon tissues. We also show that the therapeutic peptide TVP may reverse this UPR suppression and relieve ER stress leading to animal survival. Our study, along with the current literature, shows that Shiga-like toxin induced ER stress is a promising area for future study.

IntroductionEndocrine disrupting chemicals (EDCs) are agents, found either singly or used in mixtures, that disrupt the endocrine system or its production of hormones and may cause adverse effects in the exposed individuals or populations and, in the case of pregnant women, their offspring. It is presumed that maternal thyroid hormones contribute to fetal brain development. The EDCs that could impair maternal thyroid hormone function might result in subtle deficits in neurodevelopmental outcomes in children. Polychlorinated biphenyls (PCBs) are a class of EDCs produced in the past century and are still found in the environment. This dissertation aims to explore, elaborate, and embellish the associations between prenatal exposure to PCBs, the metabolites associated with PCBs, and child cognitive and motor development. The next aim of this dissertation is to explore the associations between prenatal exposure to PCBs, the metabolites associated with PCBs, and maternal thyroid hormones. Five chapters are included in this dissertation: First, an introduction to PCBs and specific aims; second, a systematic review of the literature, including the studies where prenatal exposure to PCBs or the hydroxylated PCB metabolites (OH-PCBs) and their relationship with various domains of children's neurodevelopment were investigated; third, an empirical study of exploring the associations between prenatal exposure to a mixture of PCB, OH-PCBs and children cognitive and motor skills measured in different ages; fourth, an empirical study of investigating the associations between prenatal exposure to a mixture of PCB, OH-PCBs and maternal thyroid hormones with potential for investigating the mediation of possible associations observed in the third chapter by maternal thyroid hormone concentrations; fifth, a discussion of the findings, implications for public health research, and practice and conclusions. Materials and Methods The systematic review included peer-reviewed studies indexed in several repositories (N=71 studies) from the inception of the repositories. This systematic review of the studies measured the PCBs directly in prenatal or immediately after delivery in maternal serum, cord blood, placenta, or breast milk. The empirical studies used the data available on mothers and children from "Child Health and Development Studies." Eleven PCB congeners and five OH-PCB metabolites were measured in the maternal serum post-partum among a random subset of the participants. I used a mixture analysis, Bayesian Kernel Machine Regression (BKMR), and generalized linear models (GLM) to assess the association between concentrations of PCB congeners and OH-PCB metabolites measured three days post-partum and children's cognitive and motor skills scores at age five, and cognitive skills measured at a follow-up at ages 9-11. BKMR and GLM were also used to explore the associations between PCB congeners and OH-PCB metabolites and maternal free thyroxine (FT4) and thyroid-stimulating hormone (TSH). Results The systematic review found that prenatal exposure to PCBs was possibly associated with poor cognitive development and poor attention in early and middle childhood. The evidence regarding motor development, behavior, and other neurodevelopmental outcomes were not conclusive at any stage in childhood. There was an indication for sex-specific associations with worse cognition and attention scores among boys. There was also evidence in individual studies regarding the possible association between prenatal exposure to OH-PCBs and neurodevelopmental outcomes. There were significant differences between the studies in markers of exposure, exposure assessment timing, outcome assessment, and the methodological approaches to assess the association. In the second empirical study, I found that in the crude and adjusted BKMR models among the OH-PCB metabolites, cognitive skills test scores at age five increased with each decile increase in the mixture of OH-PCB metabolites compared to when all of the metabolites were fixed at their 50th percentile among all the children and the boys and girls. These associations were largely driven by OH-PCB153 and OH-PCB146, metabolites associated with the congener PCB153. In the third empirical study, among OH-PCB metabolites attributed to PCB congeners, I observed suggestive evidence for a positive association with maternal FT4, particularly in the highest percentile of exposure to overall OH-PCB metabolites. I did not observe an association between exposure to the mixture of PCB congeners and Maternal FT4 or TSH. Nonetheless, the overall patterns suggested a positive association between exposure to PCB congeners and maternal FT4.No associations of the overall mixture of PCBs and OH-PCB metabolites were observed with maternal TSH concentrations in the BKMR models. Nonetheless, in single metabolite risk estimates, I found that 3’-OH-PCB138 was positively associated with maternal TSH values when the 3’-OH-PCB138 was fixed at its 90th percentile compared to when it was in its 10th percentile, fixing all the other congeners in their 10th, 50th, and 90th percentiles. Finally, there was an indication of a possible antagonistic interaction between 4-OH-PCB107 and 3’-OH-PCB-138 in the association of OH-PCBs with maternal FT4 such that 3’-OH-PCB138 tended to have a positive association with maternal FT4 when 4-OH-PCB107 was fixed in the 90th percentile and tended to have a negative association with maternal FT4 when 4-OH-PCB107 was fixed in their 10th percentile when all the other metabolites were fixed in their median value. Conclusions There were patterns of positive associations of OH-PCB metabolites with the children’s cognitive outcomes and maternal FT4 and maternal TSH. Nonetheless, the observed associations were weak in magnitude, often including the null value. The BKMR allowed me to explore and investigate the interactions between different components of the mixture and the overall mixture effect on the outcomes. My dissertation indicated the necessity of a holistic approach to address the impact of exposure to environmental toxins, specifically due to the detected findings regarding interactions between OH-PCB metabolites.

Les récepteurs couplés aux protéines G (RCPG) sont des protéines membranaires impliquées dans la communication entre cellules via des messagers circulants (hormones, neurotransmetteurs) ainsi que dans la perception de notre environnement (vision, odorat, goût). Ils sont essentiels à de nombreuses fonctions physiologiques vitales (cardiaques,respiratoires...) et comportementales (relations sociales et affectives) et sont donc une cible thérapeutique de choix pour la découverte de nouveaux médicaments.Au sein de l'équipe Canaux, de l’Institut de Biologie Structurale, un biocapteur original a été créé se basant sur la fusion de ces RCPG avec un canal ionique (Kir6.2) appelé Ion Channel-Coupled Receptor (ICCR). Les changements conformationnels du récepteur induit par son activité (fixation de ligand, activation des protéines G) sonttraduits par le canal ionique en courant électrique aisément détectable par des techniques électrophysiologiques. Cette nouvelle génération de biocapteurs permet d'étudier en temps réel l’activité des RCPG par des techniques électrophysiologiques très sensibles.Le travail de thèse s’est principalement focalisé sur l’étude du récepteur de l’ocytocine (OXTR) impliqué dans l’accouchement, l’allaitement et le lien social. La technologie ICCR a été utilisée pour trois des projets de cette thèse. Le premier avait pour but l’étude des mécanismes moléculaires de la dépendance au cholestérol du récepteur del’ocytocine, et a ainsi permis d’identifier un nouveau mécanisme de régulation allostérique entre le cholestérol et la fixation des ligands. Un second projet a porté sur l’utilisation de ce biocapteur pour identifier de nouveaux types de ligands, plus spécifiques de certaines voies intracellulaires, appelés ligands biaisés. Enfin, un troisième projet a mis en relief l’effet de certains composés environnementaux sur les RCPG et a permis de mettre en avant de nouveaux récepteurs ciblés par ce type de composés.Pour terminer, un projet parallèle s’est porté sur l'étude de la formation de pores par des protéines bactériennes dépendantes des RCPG. Il s’agit des « pore-forming toxins » (PFTs) de la famille des hémolysines gamma, produitespar un des pathogènes humains les plus virulents, Staphylococcus aureus. Certaines de ces toxines sont capables de sefixer sur des RCPG très spécifiques, les récepteurs aux chimiokines, et ont donc un rôle important dans les infections virales et dans certaines pathologies cancéreuses. Les travaux ont notamment permis d’obtenir des informations nouvelles sur le mécanisme d’insertion de ces pores dans la membrane. / G protein-coupled receptors (GPCRs) are membrane proteins involved in communication between cells via circulatingmessengers (hormones, neurotransmitters) as well as in the perception of the environment (vision, smell, taste). Theyare essential for many physiological functions (cardiac, respiratory...) and behavioral (social and emotional responses)and therefore represent interesting therapeutic targets.Within the Channels team, at the Institute of Structural Biology, an original biosensor was created, based onthe fusion of a GPCR to an ion channel (Kir6.2), called an Ion Channel-Coupled Receptor (ICCR). Conformationalchanges of the receptor induced by its activity (ligand binding, activation of G proteins) are directly transmitted to theion channel and allow the generation of an electrical signal easily detectable by electrophysiological techniques. Thesenew biosensors are powerful tools to study GPCR activity in real time.The main focus of the thesis was the study of the oxytocin receptor (OXTR), involved in childbirth,breastfeeding and social bonding. ICCR technology has been used for three projects during the thesis. The first aimedat studying the molecular mechanisms of cholesterol dependency of the oxytocin receptor and allowed theidentification of a new allosteric regulation mechanism between cholesterol and the ligand binding. A second projectfocused on the use of this biosensor to identify new types of ligands, selective to certain intracellular pathways, calledbiased ligands. Finally, a third project highlighted the effect of certain compounds, known as endocrine disruptors, onGPCRs. Endocrine disruptors are environmental pollutants which have potentially harmful effects on human health.Finally, a parallel project was dedicated to the study of pore formation by GPCR-dependent bacterial toxins.These proteins are called pore-forming toxins (PFTs), from the gamma hemolysin family and are produced by one ofthe most virulent human pathogens Staphylococcus aureus. Some of these toxins are able to bind very specifically tocertain GPCRs, members of the chemokine receptor family. They therefore play a vital role in numerous viralinfections and in some cancerous pathologies. New information concerning the mechanism of membrane insertion ofthese toxins during pore formation was discovered during this work.

No description available.

No description available.

The type VI secretion system (T6SS) is a protein export pathway that mediates competition between Gram-negative bacteria by facilitating the injection of toxic effector proteins from attacking cells into target cells. To function properly, many T6SSs require at least one protein that possesses a proline-alanine-alanine-arginine (PAAR) domain. These PAAR domains are often found within large, multi-domain effectors that possess additional N- and C-terminal extension domains whose function in type VI secretion is not well understood. The work described herein uncovers the function of these accessory domains across multiple PAAR-containing effectors. First, I demonstrated that thousands of PAAR effectors possess N-terminal transmembrane domains (TMDs) and that these effectors require a family of molecular chaperones for stability in the cell prior to their export by the T6SS. Our findings are corroborated by co-crystal structures of chaperones in complex with the TMDs of their cognate effectors, capturing the first high-resolution structural snapshots of T6SS chaperone-effector interactions. Second, I characterize a previously undescribed prePAAR effector named Tas1. My work shows that the C-terminus of Tas1 possesses a toxin domain that pyrophosphorylates ADP and ATP to synthesize the nucleotides adenosine penta- and tetraphosphate (hereafter referred to as (p)ppApp). Delivery of Tas1 into competitor cells drives the rapid accumulation of (p)ppApp, depletion of ADP and ATP, and widespread dysregulation of essential metabolic pathways, resulting in target cell death. These findings reveal a new mechanism of interbacterial antagonism, the first characterization of a (p)ppApp synthetase and the first demonstration of a role for (p)ppApp in bacterial physiology. TMD- and toxin-containing PAAR proteins constitute a large family of over 6,000 T6SS effectors found in Gram-negative bacteria. My work on these proteins has uncovered that different regions found within effectors have distinct roles in trafficking between bacterial cells and in the growth inhibition of the target cell. / Dissertation / Doctor of Philosophy (PhD) / Bacteria constantly compete with their neighbours for resources and space. The type VI secretion system is a protein complex that facilitates competition between Gram-negative bacteria by facilitating the injection of protein toxins, also known as effectors, from attacking cells into target cells. In this work, I characterize several members of a large family of membrane protein effectors. First, I showed that these effectors require a novel family of chaperone proteins for stability and recruitment to the type VI secretion system apparatus. Second, I characterized the growth-inhibitory properties of one of these effectors in-depth and showed that it possesses a toxin domain that depletes the essential nucleotides ATP and ADP in target cells by synthesizing the nucleotides adenosine penta- and tetraphosphate, (p)ppApp. Together, these studies revealed a new mechanism for the intercellular delivery of membrane protein toxins and uncovered the first known physiological role of a (p)ppApp-synthesizing enzyme in bacteria.

The thermotolerance, an adaptive phenomenon that is accompanied by the phenotypic plasticity which is the adjustment of physiology, biochemistry and metabolism of every cellular function by the hidden mechanism. Mitochondrion, the powerhouse of the cell that determines the functional integrity of every cellular homeostasis and functional phycological processes should provide its association in regulating the thermotolerance as well. This study assessed the mitochondrial function in regulating and determining the limit of thermo tolerance in the Daphnia magna of different geographical regions of the world, mainly sub grouped as temperature tolerant clones (IL) and temperature sensitive clones (GB). The acclimation effects or the adjustment of the preexisting biological properties help the organism adjust its biological processes to the changing habitat to maintain the cellular functional integrity. The clonal divergence as well as the acclimation show a clear pattern in limiting the thermotolerance and the prediction is the temperature tolerant clones should show higher adjustment of the mitochondrial function than temperature sensitive ones. We hypothesize that the damage in the mitochondrial membrane integrity by different mito-toxins should decrease the heat tolerance by decreasing the membrane potential and fluidity. The integrated mitochondrial function was assessed in acclimated clones by using the molecular studies as well as observation of behavioral and phenotypic plasticity. Due to the specific effects of each mito-toxins (CCCP, NaN3 and DNP) on different complexes (I-IV and ATP synthase) in ETC, we determined the mitochondrial membrane integrity by the Rhodamine 123 alongside with the lactate assay for measuring the mitochondrial integrity. Among all these three mito-toxins, CCCP show significant effect on limiting the heat tolerance. The lower lactate accumulation was observed in the temperature-tolerant clones acclimated in cold temperatures (18°C) which indicates the higher mitochondrial adjustment than the temperature sensitive clones. The concluding remark is that thermal tolerance is determined by the adjustment of mitochondrial function which accompanied with the adjustment to the mitochondrial respiration as well as the adjustment to membrane potential and fluidity.

A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A₂ (PLA₂). Lipid thermal phase transition was found to have a dramatic effect on PLA₂ activity. It was also established that although membrane binding and insertion was essential for of PLA₂ activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA₂ secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA₂. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.