Lightning Talks
Ryan Benke; Purdue University, Biochemistry
Using Metabolomics and Association Genetics to Map Lesion Mimic Mutants
Plant mutants that form lesions and/or undergo spontaneous cell death in the absence of pathogens or stress have been isolated from a wide variety of genetic systems. These mutants form spontaneous lesions due to constitutively active disease signaling or defects in metabolism that result in cell death. Of the more than 50 maize lesion-forming mutants, the etiologies of only four of these are known. Using untargeted metabolite profiling we have explored the metabolic consequences of 24 maize lesion-forming mutants and compared them to mutants with constitutively activated hypersensitive-response (Rp1-D21) and defects in chlorophyll metabolism (e.g. Oy1-N1989). Clustering on metabolite abundances clearly indicates a metabolic syndrome similar to the HR-signaling mutant Rp1-D21 in a subset of these 24 mutants. The dominant mutant Les10 exhibits similar shifts in metabolite levels, including predicted defense metabolites (e.g. salicylic acid) and chlorophyll breakdown products, as observed in Rp1-D21. The Les10 gene was previously mapped to chromosome 2L using translocation stocks and mutant linkage. We used genome-wide association to explore whether natural variation encoded suppressor or enhancer alleles affecting Les10 phenotypic severity. An F1-hybrid population was generated by crossing an association panel by Les10/+ pollen parents and phenotyped for lesion severity, chlorophyll content, and plant height. The polymorphisms with the strongest association with these traits mapped to the region encoding Les10, suggesting that a cis-QTL affecting the Les10 locus itself may be responsible for the largest genetic effect on the mutant phenotype in this population. The top SNP is near an ankyrin-domain containing gene similar to Arabidopsis ACCELERATED CELL DEATH6. A dominant allele of acd6 in Arabidopsis undergoes spontaneous cell death, suggesting this maize gene is a good gene candidate for Les10.
Elias Beretta; Purdue University, Chemistry
The Use of Dimeric Substrates as Prodrugs and Inhibitors of P-glycoprotein
The blood brain-barrier (BBB) serves as a protective barrier that limits the entry of many agents into the brain and poses a serious challenge for delivery of therapeutics to the brain. One component of the BBB that is responsible for this difficulty is the ATP Binding Cassette (ABC) transporter P-glycoprotein (P-gp), which is expressed in the luminal membrane of brain capillary endothelial cells. P-gp uses the energy of ATP hydrolysis to actively remove a variety of compounds from the membrane and has a polyvalent substrate binding site. Many brain therapeutics used to treat a number of disorders are substrates of P-gp and thus, accumulation of these drugs in the brain is limited by its expression. To take advantage of the polyvalency of the binding site, we synthesized and characterized of a library of dimeric prodrug inhibitors of P-gp using the glioblastoma multiforme chemotherapeutic, temozolomide. In addition to inhibiting Pgp, these dimers are also designed to act as prodrugs and release the therapeutic after crossing the BBB. Using the known substrate quinine, a library of temozolomide-quinine heterodimers with varying linker lengths was generated. Temozolomide and quinine alone were both found to not be inhibitors of P-gp, even at high concentrations (> 100 μM) . On the other hand, the heterodimers were shown to be potent inhibitors of P-gp mediated efflux at low micromolar concentrations. Initial studies with our temozolomide-quinine heterodimer library showed inhibition ranging from 1-10 μM in P-gp expressing MCF-7 and 12D7 cell lines. Our results demonstrated the potential of these temozolomide-quinine heterodimers to act as inhibitors of P-gp, as well as serve as prodrugs for delivery of temozolomide across the blood brain barrier. We anticipate that our dimers will improve the efficacy of temozolomide treatment of glioblastoma multiforme by mitigating the effect of P-gp efflux.
Ariana Cardillo; Purdue University, Chemistry
The Role of Methylation in the Regulation of KRas4B Distribution and Activity
Isoprenylcysteine carboxyl methyltrasferase (ICMT) is an endoplasmic reticulum-restricted integral membrane protein responsible for a post-translational modification (PTM) at the C-terminus of CaaX proteins. Over 300 CaaX proteins have been identified, with the four Ras isoforms being the most well-studied. When fully modified, these small GTPases are tethered to the plasma membrane and regulate the relay of extracellular, mitogenic signals downstream through pathways that lead to cell growth and differentiation. KRas is mutated in over 90% of all pancreatic cancers making it a desirable target for chemotherapeutic treatment. However, its reputation for being “undruggable” has led to the development of interventions that target upstream posttranslational modification machinery. Some evidence suggests that blocking correct KRas localization through ICMT inhibition may be an alternate therapeutic avenue. Paradoxically, some KRas may still traffic to the plasma membrane in the absence of active ICMT. Therefore, the relationship between KRas methylation, membrane targeting, and signal propagation remain unclear, particularly in the cases of specific KRas mutations. Using a panel of isogenic KRas4B cell lines with and without KRas isoform protein overexpression, we are measuring the distribution of Ras at various cellular locations and their activity upon ICMT inhibition. Complimentary imaging-based biophysical and biochemical approaches are expected to aid in our understanding of the role of methylation not only for KRas but for the other hundreds of CaaX proteins involved in disease pathways.
Austin Cool; The Ohio State University, Chemistry & Biochemistry
Computational Methods Elucidate Consequences of Mutations and Post-Translational Modifications on Troponin I Effective Concentration to Troponin C
Ca2+ binding to cardiac troponin C (cTnC) causes a conformational shift that exposes a hydrophobic patch (cTnCHP) for binding of the cTnI switch peptide (cTnISP), ultimately resulting in contraction of the heart. The inhibitory peptide (cTnIIP), attached at the N-terminal end of the cTnISP, serves as a tether for the cTnISP to the rest of the troponin complex. Due to this tethered nature, the cTnISP remains within proximity of the hydrophobic patch region, resulting in the cTnCHP experiencing an elevated “effective concentration” of the cTnISP. Mutations to the cTnIIP region have been hypothesized to cause disease by effecting the ability of the cTnISP to ‘find’ the hydrophobic patch, resulting in alterations to the heart’s ability to contract normally. We tested this hypothesis using molecular dynamics (MD) simulations of the troponin complex using a model that contained all three subunits of troponin: C, I, and T (cTnT). We developed methods that allowed us to quantitatively measure the effective concentration of the cTnISP from the simulations. A significant reduction in cTnISP effective concentration was observed when the cTnIIP was removed from the system, showcasing the importance of a tethered cTnISP. Through accelerated MD methods, we proposed the minimum effective concentration of a tethered cTnISP to be approximately 21mM. Modification of the cTnIIP via PKC mediated phosphorylation of T143 was shown to significantly increase the estimated effective concentration of cTnISP, help the cTnISP find the cTnCHP faster, and maintain the relative shape of the cTnIIP when compared to the native model. All of this data indicates that pT143 may be able to help promote binding of cTnISP to the cTnCHP. We then tested six mutations within the cTnIIP region that are known cTnC Ca2+ sensitizing mutations, and have been linked with cardiomyopathy. We did not observe a significant reduction in effective concentration upon introduction of these mutations, however we did observe increased variability in the flexibility and dynamics of the cTnIIP region when compared to native. Our observations led us to hypothesize that the mechanism by which these cardiomyopathic mutations effect Ca2+ sensitivity is by altering the off rate of cTnISP from the hydrophobic patch.
Barrett Davis; Purdue University, Biomedical Engineering
Computational Model of PDZ-Binding Competition and AMPA Receptor Insertion in the Postsynaptic Density (PSD)
Neurodevelopmental disorders (NDD), including Autism Spectrum Disorders(ASD) are estimated to affect between 2-5% of all children, with an associated US economic burden of over $100B.ThoughNDDs often stem from de novo mutations, mounting evidence has identified some common neuronal pathophysiology in trans-and postsynaptic scaffolding proteins including neuroligin (NLGN), leucine-rich repeat transmembrane protein(LRRTM) and post-synaptic density protein 95 (PSD-95).This “common pool” interacts through shared PDZ-binding motifs and is essential to the fine-tuning of long-term synaptic plasticity. At the molecular level, this tuning process is modulated by both glutamatergic receptor availability (i.e., AMPAR)and molecular competition for PDZ-binding sites which then anchor these receptors into the post synaptic density (PSD). While knockouts of PDZ-binding partners like NLGN and LRRTM have been shown to strongly bias synapses towards long-term potentiation(LTP), at present we lack adequate mechanistic models to describe the transient nature of receptor availability and scaffolding protein competition during calcium stimulation. Therefore, building from previous work in our lab, here we present a compartmentalized, rule-based model for activity-dependent insertion of AMPARs into the post-synaptic density. We further extend this work by simulating in vitro models of disease and characterizing the effects of simulated genetic and pharmaceutical therapies.
Muriel Eaton; Purdue University, MCMP
Substantial reduction of Scn2a expression renders behavioral abnormalities in mice indicative of autism spectrum disorder
Recent exome sequencing studies have discovered a strong correlation between the voltage-gated sodium ion channel Nav1.2 (encoded by gene Scn2a) and autism spectrum disorder (ASD), as well as other neurodevelopmental disorders. Nav1.2, together with Nav1.1 and Nav1.6, are major sodium channels expressed in the central nervous system playing major roles in contributing to neuronal excitability. Homozygous knockout of Scn2a -/- is perinatal lethal. Heterozygous Scn2a +/- mice display some mild abnormalities but do not seem to recapitulate severe disease phenotypes. To further explore the relationship between the dysfunction of Scn2a, ASD, and its comorbidities, our lab acquired a mouse model in which the expression of Scn2a is substantially reduced. We are currently conducting a behavioral battery to assess the phenotype of these mice including sociability, anxiety, and innate behavior. We plan to test both males and females to determine if there are sex differences. The data we obtain will justify that these mice can be used as a model to study severe ASD associated with Scn2a loss-of-function and its related comorbidities.
Isaac Fisher; Purdue University, Chemistry
Towards Determination of the Activation Mechanism of PLCβ by Gβγ
G protein-coupled receptors (GPCRs) regulate diverse physiological processes in health and disease via activation and release of the heterotrimeric G protein subunits, Gα and Gβγ. Gα and Gβγ activate effector enzymes, promoting second messenger production. One such effector enzyme is phospholipase C β (PLCβ). PLCβ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) at the plasma membrane to generate membrane-bound diacylglycerol (DAG) and cytosolic inositol-1,4,5 trisphosphate (IP3). These second messengers activate protein kinase C (PKC) and increase intracellular Ca2+, respectively, which allows PLCβ to act on numerous cellular process downstream of G-protein activation; however, aberrant PLC signaling has been implicated in numerous diseases. Indeed, targeting G-protein-PLC interactions has been shown to have therapeutic potential in multiple physiologies, such as inflammation, cardiac hypertrophy, opioid analgesia, and cancer. PLCβ enzymes have modest basal activity and are potently activated by Gαq released by Gq-GPCRs. PLCβ2 and PLCβ3 are also regulated by Gβγ, released by Gi-GPCRs. While much has been learned about how Gαq binds to and activates PLCβ, there is no consensus as to the mechanism of Gβγ-mediated activation. Here, we discuss the potential conformational changes that PLCβ undergoes when activated by G-proteins on lipid membranes, as well as current attempts at structure determination of Gβγ-PLCβ complexes, in the presence or absence of membrane memetics, by cryo-electron microscopy (cryo-EM). We hope to utilize this structural information in the future development of chemical probes to selectively target specific conformations of PLCβ.
Elizabeth Garland-Kuntz; Purdue University, Chemistry
Structural Insights into PLCε: Discovery of an Integrated RA1 Domain and Novel Regulatory Elements
Phospholipase Cε (PLC) enzymes hydrolyze phosphatidylinositol lipids at cellular membranes to produce inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers increase intracellular Ca2+ and activate protein kinase C (PKC), allowing PLCε to contribute to numerous processes in response to G protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) activation. However, aberrant PLCε activity leads to cardiac hypertrophy and upregulation of oncogenic and inflammatory signaling pathways. PLCε shares a conserved core with other PLC enzymes but contains additional regulatory domains at its N- and C-terminus that contribute to autoregulation, membrane association, and G protein-dependent activation. However, the large size and conformational heterogeneity of this enzyme present formidable hurdles for high-resolution structure determination. Here, we present the 2.7 Å structure of a catalytically active PLCε variant, spanning the EF3-RA1 domains, providing the first structural insights into this subfamily. This structure reveals that the C-terminal Ras association (RA) 1 domain forms extensive intramolecular interactions with the rest of the lipase. Using biochemical and cell-based assays, we also identified two PLCε regulatory elements: a highly conserved amphipathic helix within the catalytic domain, and the linker connecting the RA1 domain to the core. We are now pursuing high-resolution structures of larger fragments of PLCε using domain specific Fabs, with the goal of using structure-guided drug discovery to identify modulators of this critical enzyme.
Samadhi Kulathunga; Purdue University, Chemistry
Preliminary attempt of the validation of SULT2B1b specific Coupled-enzyme assay using DESI-MS
According to the latest statistics by the American Cancer Society, prostate cancer is the most frequently diagnosed and the second leading cause of cancer death in American men estimating more than 200,000 new cases for 2021. These statistics emphasize the importance of identifying new drug targets and developing novel therapeutics for this deadly disease. One specific drug target that has been found to have abnormally higher levels in prostate cancer cell lines is sulfotransferase 2B1b (SULT2B1b), an enzyme that sulfonates 3β-hydroxysteroids to 3β-hydroxysteroid sulfates. This study investigates the biological function of human SULT2B1b using in-vitro kinetic characterization in an effort to develop novel therapeutics for prostate cancer treatment. To date, there has been no published work on the complete kinetic characterization profile of SULT2B1b with all of its 3β-hydroxysteroid substrates (i.e. cholesterol, pregnenolone, DHEA) and there are significant differences among the published kinetic data which were determined using radio labeled assay as well. Hence, the major limitation in developing inhibitors for SULT2B1b is the lack of its proper biochemical characterization. To address this gap in knowledge, we have developed a continuous, fluorescent based, coupled-enzyme assay for in-vitro characterization of SULT2B1b and to screen small-molecule inhibitors. In this assay, SULT2B1b activity is indirectly measured by correlating the reduction of a 3β-hydroxysteroid substrate to the formation of fluorescent molecules. Using this coupled-enzyme assay, we could determine various kinetic parameters of SULT2B1b such as substrate affinity (Km) and turn over number (kcat). One limitation of this assay is that; it indirectly monitors the SULT2B1b activity by utilizing another sulfotransferase, SULT1A1, as the coupled-enzyme which occasionally causes false positive results in inhibition studies. Therefore, it is essential to validate the primary kinetic and inhibition results using a secondary assay. This study is a preliminary attempt of validating our coupled-enzyme assay using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) where we can directly measure SULT2B1b activity by monitoring the formation of 3β-hydroxysteroid sulfates or catalytic products. Preliminary results of this study demonstrated the comparative nature of the two methods showing minimal differences among kinetic parameters. To further validate coupled-enzyme assay, inhibition studies will be carried out with DESI-MS using small molecule-inhibitors. This study will further the drug discovery field of prostate cancer since the validation of an “in-vitro target assay” is the fundamental step to enabling successful drug discovery pipeline against SULT2B1b.
Chennan Li; Purdue University, Biological Sciences
Loss of ARPC3 drives anchorage independent growth in KRAS;TP53-mutated human bronchial epithelial cells
Lung cancer is by far the leading cause of cancer-related deaths. KRAS and TP53 are two of the most frequently mutated genes in lung cancer, and their mutations are well recognized as drivers of tumorigenesis. Directly targeting these drivers still remains a therapeutic challenge. Instead, targeting genes that potentiate KRAS/TP53-driven tumorigenesis is a rational alternative strategy. Therefore, we hypothesized that loss of certain protein-coding or microRNA genes, can drive cellular transformation of a non-transformed KRAS;TP53-mutated human bronchial epithelial cell line (HBEC-KP). To address this hypothesis, a forward genomic selection experiment was performed using CRISPR/Cas9. Anchorage-independent (AI) growth, a hallmark of cancer, was selected as a phenotypic readout for one experiment. Wildtype HBEC-KP cells were incapable of AI growth. Only certain mutants were capable of promoting colony formation in soft agar. These clones were isolated and the integrated sgRNAs were identified. Through this approach, we discovered that loss of ARPC3 drives AI growth in the HBEC-KP cells. Genetic rescue by overexpression of ARPC3 partially reverts the AI phenotype. In contrast to the growth-suppressive role of ARPC3 in soft agar, loss of ARPC3 results in defective cell migration, invasion, and clonal expansion in the HBEC-KP cells. This suggests that ARPC3 is essential for cell motility and proliferation, which is consistent with other reports. Based on this evidence, we hypothesize that loss of ARPC3 causes AI growth through reducing detachment-induced cell death, rather than increasing cell proliferation. To test this hypothesis, HBEC-KP and HBEC-KP;ARPC3-knockout cells were pre-treated in a poly-HEMA-coated suspension plate, and fluorescent propidium iodide (PI) was then used to discriminate between live and dead cells. Through flow cytometry analysis, HBEC-KP;ARPC3-knockout cells show significantly reduced cell death following pre-treatment of cells in suspension compared to the HBEC-KP cells, whereas no significant difference in cell death was observed without pre-treatment. Extensive mechanistic studies are being conducted to determine how loss of ARPC3 causes detachment-induced cell death and ultimately, AI growth in the context of mutated KRAS;TP53. Clinical relevance is also being closely examined to further understand the role of ARPC3 in human cancer.
Akansha Maheshwari; Purdue University, Chemistry
Elucidation of the biochemical and structural aspects of yeast Icmt (Ste14) along with the development of ICMT inhibitors for Ras-driven Cancer
CaaX proteins are proteins with a CaaX box at the end of their C terminus (C: cysteine, A: Aliphatic residues and X: one of the several amino acids.) This CaaX box which provides an indication that the protein needs to undergo 3 sequential post translational modification which can then help in its localization to the plasma membrane from where it can carry out its varied cellular function. The three modifications include: prenylation of the cysteine residues, endoproteolysis of the -aaX residues, and lastly carboxylmethylesterification of the isoprenylated cysteine residues by an integral ER membrane protein ICMT. One of the most studied CaaX protein, Ras has been implicated in numerous cancer cases. More specifically pancreatic cancer, which has no effective cure till date, has around 90% cases which involve a mutated Ras isoform KRas. Thus, studies are underway to inhibit the enzyme involved in three post translational modifications to thus promote mislocalization and inhibition of Ras In such cancer cases. My research is to more specifically study the biochemical and structural aspect of yeast ICMT, Ste14, to gain more understanding of how Ste14 carries out methylesterification of Ras, followed by plasma membrane localization and which residues are important for function. I also aim to study the effect of developed ICMT inhibitors on mislocalization of Ras as well as on ICMT enzymatic activity. These inhibitors could serve as potential chemotherapeutic drugs for Ras-driven cancer. My aim to approach this research is to run radioactivity assays to test the efficiency of ICMT inhibitors so as to evaluate the IC50s of these inhibitors so as to conclude which inhibitor could be potentially used as a therapeutic tool for Ras driven cancer such as pancreatic cancer.
Vinay Menon; Purdue University, Chemistry
The Biophysical Properties of the Broad-Spectrum Antimicrobial Peptide, P14LRR
According to the CDC, methicillin resistant Staphylococcus aureus (MRSA) infections result in over 300,000 hospitalizations and $1.7 billion in healthcare costs annually in the United States. This is exacerbated by the ability of some pathogenic bacteria to sequester within mammalian cells, especially macrophages. To further compound this problem, current commercially available therapies are unable to effectively penetrate mammalian cells and accumulate in therapeutic doses. The broad-spectrum anti-microbial peptide (AMP), P14LRR, has demonstrated good activity against gram-positive and gram-negative bacteria (MIC ≈ 16 µM) and has been shown to eradicate up to 90% of intracellular bacteria. The anti-bacterial target of P14LRR in S. aureus has been identified as the 47 kDa glycolytic enzyme, Enolase. Herein we present a biophysical characterization of the interaction between P14LRR and Enolase, including the dissociation constant (Kd) and thermodynamic constants (∆G, ∆H, and ∆S). These data will aid in the further improvement of our peptides by providing a foundation that we can compare subsequent generations and derivatives to.
Kaushik Muralidharan; Purdue University, Biological Sciences
Understanding molecular mechanism of PLCε regulation by small G-proteins
Cardiovascular disease is the leading cause of death in the United States. The phospholipase C (PLC) family of enzymes catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control cellular function and behavior. The PLCε subfamily is required for normal cardiovascular function, where it is regulated through direct interactions with the RhoA and Rap1A small GTPases, which are activated in response to G protein-coupled receptor (GPCR stimulation). RhoA activates PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the domains of PLCε involved in G protein binding and activation, and translocation to different subcellular membranes is largely unknown. In this work, we use cell-based activity assays, epifluorescence, and confocal microscopy to identify the domains of PLCε involved in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Our preliminary studies demonstrate that the unique N- and C-terminal regulatory domains of PLCε dictate its location within the cell and contribute differently to basal and G protein-dependent activity. These studies will provide needed insights into the regulation and localization of PLCε in cells, which is critical for its roles in cardiovascular function.
Vaani Ohri; Purdue University, Biological Sciences
Investigating RhoA-Dependent Regulation of Phospholipase Cε in Cardiovascular Disease
Cardiovascular diseases are the leading cause of death in the United States. A leading contributor to this alarming statistic is cardiac hypertrophy, characterized by an abnormal enlargement of the heart muscle that is triggered, in part, by phospholipase C ε (PLCε) overexpression. The PLCε enzyme is responsible for hydrolyzing membrane phosphatidyl inositol 4,5- bisphosphate (PIP2) into two important secondary messenger molecules: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG activates protein kinase C (PKC), while IP3 binds to the IP3 receptor present on the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) to increase intracellular Ca2+. Together, these molecules activate a myriad of signaling pathways that instigate cardiac contractility and hypertrophy. Interestingly, even though PLCε hyperactivation induces cardiac hypertrophy, PLCε activation mediated by the RhoA small GTPase is actually cardioprotective, especially in the case of ischemia/reperfusion injuries. In vitro and cell-based activity assays have shown that an insertion within the catalytic domain of PLCε, referred to as the Y box, is required for RhoA-mediated activation of PLCε. However, this element is dispensable for GTPase binding, and the site of RhoA binding is not known. We hypothesize that RhoA binds to the PLCε catalytic core, and promotes a conformational change in PLCε that allosterically activates the lipase. In this study, we will use biochemical activity and binding assays to identify the smallest PLCε fragment that maintains basal activity and is activated by RhoA. X-ray crystallography will be used in parallel to solve the structure of this fragment in complex with RhoA. It is also possible that RhoA also spatially regulates PLCε at the membrane, helping to anchor the lipase active site to the plasma membrane. We will use single molecule tracking studies on supported lipid bilayers to understand the membrane kinetics involved in the signaling pathway of RhoA-mediated PLCε activation. Together, these studies will elucidate the roles that RhoA plays in translocating and activating PLCε at the plasma membrane, and protecting cardiovascular function.
Kadidia Samassekou; Purdue University, Chemistry
Structural and Functional Insights into Scaffolding of PLCε by mAKAP
Muscle-specific A-kinase anchoring protein (mAKAP) is a large, conserved scaffolding protein that is exclusively expressed in striated muscle like the heart, where it is involved in cytokine and adrenergic-induced hypertrophy. In cardiomyocytes, mAKAP is found on the outer membrane of the nuclear envelope, where it serves as a scaffold for proteins that are involved in cardiac hypertrophy, including the enzyme phospholipase C ε (PLCe). This enzyme cleaves phosphatidylinositol phosphates (PIP2 and PI4P) into the second messengers inositol phosphates (IP3 and IP2) and diacylglycerol (DAG). An increase in IP3 stimulates the release of Ca2+ from intracellular stores, while DAG activates protein kinase C (PKC). The heart relies on highly regulated calcium levels and PKC activation for normal contraction. Previous studies have shown that PLCe interacts directly with the first spectrin repeat SR-1 in mAKAP via its RA1 domain, and importantly, disruption of this interaction prevented agonist-induced hypertrophy in neonatal rat ventricular myocytes. We hypothesize that the interaction of mAKAP with the PLCe RA1 domain disrupts an autoinhibitory element within PLCe, increasing its basal activity. PLCe is found in the cytoplasm under basal conditions, but upon activation by the Rap1A GTPase, it is translocated to the perinuclear membrane, where this activated complex may be stabilized by mAKAP. Do mAKAP and Rap1A additively or synergically regulate PLCε activity? To answer these questions, we need to understand the mAKAP–PLCe interaction at the structural and cellular level. Towards these goals, and in collaboration with the Kossiakoff lab, we have developed antigen binding fragments (Fabs) that recognize specific domains within PLCe. These Fabs will serve as fiduciary markers for high-resolution structural studies of PLCe in complex with Rap1A and mAKAP. In parallel, we are also investigating the coordinated regulation of PLCe by these regulatory proteins using cell-based functional assays.
Arielle Selvia; Purdue University, Chemistry
The Mechanisms of PLCε Activtation by Rap1A
Phospholipase Cε (PLCε) is required for normal regulation of intracellular Ca2+ release in response to stimulation of G protein-coupled receptors (GPCRs). Changes in PLCε expression and activity are implicated in cardiac hypertrophy and heart failure, which is the leading cause of death in the United States. This disease process is driven by the Rap1A GTPase, which is activated downstream of beta adrenergic receptors. Rap1A binds to the C-terminal RA2 domain of PLCε, but how this binding activates PLCε is not known. Using a series of PLCε domain deletion variants, we investigated whether other domains in the lipase contributes to Rap1A-dependent activation. In addition to the RA2 domain, we found that the N-terminal pleckstrin homology (PH) domain and first two EF hands (EF1/2) are also required for Rap1A-dependent activation. We then used small angle X-ray scattering (SAXS) was used to investigate whether Rap1A induced conformational changes within two PLCε variants, one which retained the PH and EF1/2 domains, and one that lacked them. When the PH and EF1/2 domains are present, Rap1A stabilized an extended, less flexible conformation of the lipase. In contrast, when Rap1A binds to a PLCε variant lacking these domains, the conformational changes induced by G protein binding stabilized a more compact structure. Thus, the PLCε PH and EF1/2 domains likely contribute to Rap1A-dependent activation through an allosteric component. Studies of this molecular mechanism will ultimately provide valuable insights into the onset and progression of heart failure.
Karthik Srinivasan; Purdue University, Biological Sciences
Structural analysis of the Toc75 Potra domains from Pisum sativum
Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all green plants. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their functions. The translocon on the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components: Toc75, Toc33/34 and Toc159 with unknown stoichiometries. Our lab seeks to elucidate the structural architecture of the TOC complex to gain mechanistic insights into protein translocation in chloroplasts. Toc75 is a β-barrel membrane protein that forms the channel of the TOC translocon and our lab has previously reported a crystal structure of the N-terminal polypeptide transport-associated (POTRA) domains from Arabidopsis thaliana. In this work, we demonstrate the generation of antigen-binding fragments (Fabs) that specifically recognize the POTRA domains from Pisum sativum (pea plant). Further, we report a 2Å crystal structure of pea Potra domain 2 in complex with a Fab. We also propose a homology model for Potra domains 1 and 3 and validate the this hybrid model using size exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS). Future directions include screening more Fabs to obtain structure of all three Potra domains and probing for chaperone-like activity which has been demonstrated for the Arabidopsis variant.
Genki Terashi; Purdue University, Biological Sciences
VESPER: Global and Local Cryo-EM Map Alignment and Database Search Using Local Density Vectors
An increasing number of density maps of biological macromolecules have been determined by cryo-electron microscopy (cryo-EM). Although individual maps provide valuable structure information of macromolecules, the value of the maps in the database would be significantly raised if a computational method is available that performs accurate global and local map alignment and database search. Previously, we have developed EM-SURFER (http://kiharalab.org/em-surfer/), a web-based tool for real-time global matching and database search for EM maps. Using 3D Zernike Descriptors, an EM-SURFER search against the entire EMDB can be finished in a few seconds. On the other hand, EM-SURFER does not provide a map alignment because it uses rotation-invariant descriptors and also it only performs global matching. Here, we developed a new method VESPER (VEctor-based local SPace ElectRon density map alignment), which performs accurate global and local alignment and comparison of EM maps. The advantage of the VESPER algorithm is it matches two maps by considering local gradients represented by unit vectors, which can effectively capture underlined macromolecular structures in the maps. Thus, the directions of the vectors capture local structures embedded in the map, which turned out to be effective in obtaining accurate global and local map alignment. An alignment of maps is evaluated by a score defined as the sum of dot products of matched vectors from two maps and the best alignment with the maximum score is sought using fast Fourier transform (FFT). Compared on benchmark datasets, VESPER showed a higher accuracy in map retrieval as well as global and local map matching than other existing methods. VESPER also showed higher accuracies in both global and local EM map alignment. Our VESPER algorithm was published this year in Nature Communications.
Nicolas Varas; Indiana University, Biochemistry & Molecular Biology
A fibrillation-resistant glucagon analogue stabilized by a side-chain lactam bond
One of the main side effects of Insulin treatment in diabetic patients is the high rate of hypoglycemic episodes. Most of these episodes are manageable by sugar ingestion, but their continuous occurrence increases the probability of ischemic events, arrhythmias, neurological damage, coma, and even death. Consequently, the risk of hypoglycemia remains the most significant obstacle that prevents insulin-dependent patients from attaining acceptable metabolic control. A major restraint for advancements comes from the lack of hyperglycemic agents to counteract the exogenous insulin action. Glucagon is the natural counter-regulatory hormone, which fulfills its role by binding in a α-helical structure to glucagon receptor and subsequently stimulating hepatic glycogenolysis and gluconeogenesis. Previous clinical studies have shown a significant decrease in hypoglycemic events when using a dual-hormone, insulin/glucagon, artificial pancreas. But, despite its great potential as a drug, native glucagon has low solubility at physiological pH, it is prone to chemical degradation under acidic or basic buffers and has an intrinsic propensity to rapidly form β-amyloid fibrils, which nullify its activity and can make it cytotoxic. To address this problem our group is exploring different approaches to create novel, stable, and fibrillation-resistant glucagon analogs that could be suitable for dual-hormone pumps. One of those methods is to lock-in an α-helix turn through side-chain cyclization, particularly a lactam side-chain bond at positions i,i+4. We generated glucagon analogs by solid-phase peptide synthesis and induce side-chain cyclization using selective deprotection with subsequent amide bond formation. This strategy gave us a glucagon analogue with fibrillation-resistance for at least 11 days at 100μM in PBS buffer, under agitation and 37°C. This analog proved to be active in vitro and in vivo, even after a week of agitation at 37°C. To date, there is no glucagon analog or formulation commercially available for use in dual-hormonal pumps, and the need for an ultra-stable game-changing analog that moves beyond emergency hypoglycemia rescue is still present. We hope this lactam restrained glucagon will be the base for a new therapeutic with the goal of improving the therapeutic management of Type 1 Diabetes Mellitus.
Jacob Verburgt; Purdue University, Biological Sciences
Benchmarking of Structure Refinement Methods for Protein Complex Models
Protein structure docking is the process in which the quaternary structure of a protein complex is predicted from individual tertiary structures of the protein subunits. Protein docking is typically performed in two main steps. The subunits are first docked while keeping them rigid to form the complex, which is then followed by structural refinement. Structure refinement is crucial for a practical use of computational protein docking models, as it is aimed for correcting conformations of interacting residues and atoms at the interface. Here, we benchmarked the performance of several existing protein structure refinement methods to refine multimeric protein complexes. We show that the fraction of native contacts between subunits is by far the most straightforward metric to improve. However, backbone dependent metrics, based on the Root Mean Square Deviation (RMSD) proved more difficult to improve via refinement.
Igi Vilza; Purdue University, Chemistry
Investigating the Role of Conformational Dynamics in Phospholipase C-Membrane Interactions
Phospholipase C β (PLCβ) is an enzyme that plays a key role in cardiovascular function by hydrolyzing phosphatidylinositides (PIs) at the plasma membrane in response to activation of G protein-coupled receptors at the membrane. PLCβ activity produces the second messengers inositol triphosphate and diacylglycerol, which mobilize intracellular Ca2+ and activate protein kinase C. Dysregulation of PLCβ expression and/or activity in the heart can lead to cardiac hypertrophy and heart failure. PLCβ normally has low basal activity, and its activation is driven by the heterotrimeric G protein subunits Gβγ and Gαq. PLCβ has a conserved catalytic core, comprised of a pleckstrin homology (PH) domain, four tandem EF hands, a catalytic TIM barrel domain, a C2 domain. Immediately following the C2 domain is an autoinhibitory proximal C-terminal domain (CTD), which is connected to the distal CTD by an unconserved linker. This distal CTD is required for maximum activity and is thought to be the primary driver of membrane binding. However, how PLCβ interacts with the membrane, and whether conformational changes occur upon binding are not known. In addition, while Gβγ is an established activator of PLCβ, its binding site and mechanism of activation are unknown. Recent work from our lab and others has shown that the PH domain and first two EF hands (EF1/2) are conformationally dynamic in solution, and that restricting this flexibility eliminates Gβγ-dependent activation. In this study, we are using an intramolecular disulfide crosslinks to stabilize defined conformational states of PLCβ. These conformational variants will be assessed for their thermal stability, basal and Gβγ-stimulated activity, and their ability to interact with liposomes. These experiments will provide the first insights into how the conformational state of PLCβ contributes to binding the membrane and Gβγ, and to its activity at the membrane interface.
Ben Watson; Purdue University, Biological Sciences
α-Synuclein is a Target of FIC-mediated Adenylylation
During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic mediated adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE—alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation in vitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.