Apo Protein Research Articles

A molecular comparison of [Fe-S] cluster-based homeostasis in Escherichia coli and Pseudomonas aeruginosa.

ISC and SUF molecular systems build and transfer Fe-S cluster to cellular apo protein clients. The model Escherichia coli has both ISC and SUF and study of the interplay between the two systems established that the ISC system is the house-keeping one and SUF the stress-responding one. Unexpectedly, our recent phylogenomic analysis revealed that in contrast to E. coli (and related enterobacteria such as Salmonella), most bacteria have only one system, and, in most cases, it is SUF. Pseudomonas aeruginosa fits the general rule of having only one system but stands against the rule by having ISC. This study aims at engineering P. aeruginosa harboring E. coli systems and vice versa. Comparison of the recombinants allowed to assess the functional versatility of each system while appreciating their contribution to cellular homeostasis in different species context.

The crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase reveals active site features controlling catalytic specificity

Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generates the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial ɑ-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.

Structural insights into RNA cleavage by a novel family of bacterial RNases.

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70°and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family.

Enhancing the apo protein tyrosine phosphatase non-receptor type 2 crystal soaking strategy through inhibitor-accessible binding sites.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) has recently been recognized as a promising target for cancer immunotherapy. Despite extensive structural and functional studies of other protein tyrosine phosphatases, there is limited structural understanding of PTPN2. Currently, there are only five published PTPN2 structures and none are truly unbound due to the presence of a mutation, an inhibitor or a loop (related to crystal packing) in the active site. In this report, a novel crystal packing is revealed that resulted in a true apo PTPN2 crystal structure with an unbound active site, allowing the active site to be observed in a native apo state for the first time. Key residues related to accommodation in the active site became identifiable upon comparison with previously published PTPN2 structures. Structures of PTPN2 in complex with an established PTPN1 active-site inhibitor and an allosteric inhibitor were achieved through soaking experiments using these apo PTPN2 crystals. The increased structural understanding of apo PTPN2 and the ability to soak in inhibitors will aid the development of future PTPN2 inhibitors.

Lipoprotein(a) as underestimated cardiovascular risk factor in Russia. Time to introduce into clinical practice

An increased concentration of lipoprotein(a) (Lp(a)) is one of the main genetic residual risk factors for atherosclerotic cardiovascular diseases, as well as their complications. Lp(a) is a complex supramolecular complex in which the unique glycosylated apo(a) protein, which has a wide polymorphism, is linked by a disulfide bond to the apoB100 molecule (protein component of the low-density lipoprotein (LDL) particle). Therefore, Lp(a) is often called LDL-like particle. Unlike all other lipoproteins, Lp(a) concentration is determined genetically, almost constant throughout the life, and has wide individual and interethnic variability. As an independent risk factor, elevated Lp(a) concentrations can significantly increase global cardiovascular risk, especially in patients with other risk factors. Modern international and national guidelines recommend measuring Lp(a) concentration not only in certain categories of patients, but also in every person at least once in their life for a correct assessment of cardiovascular risk.

Investigating emodin derivatives against SARS-CoV-2 found in medicinal herbs

SARS-CoV-2 continues to mutate and circulate at alarming levels around the world and is exemplified by the emergence of the new variant JN.1. The emergence of novel SARS-CoV variants underscores the urgency for the development of new drugs. Phytochemicals, known for their safety and efficacy, have been widely used to address various ailments. This study aims at identifying the anti-SARS-CoV potential of emodin, a bioactive anthraquinone, and its derivatives. Key proteins, including hemagglutinin-esterase (HE), papain-like protease (PLpro), major protease (3CLpro), non-structural protein (nsp3) and spike protein(S) of SARS-CoV, were used as protein targets for the virtual screening of 110 emodin derivatives (ED) and remdesivir a proven antiviral drug. Among the 110 derivatives, ED21, ED25, ED5 inhibited HE, 3CLpro and S protein, respectively. ED29 inhibited both PLpro and nsp3 with high binding affinity. Similar receptor binding sites were occupied by remdesivir and emodin derivatives. These emodin derivatives are bound to similar amino acids in receptors as compared to remdesivir. Further dynamic simulations studies with ED21-HE, ED25-3CLpro, ED5-S, ED29-PLpro and ED29-nsp3 complexes showed all of them are stable with minimum root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent-accessible surface area (SASA) and radius of gyration (Rg) and are comparable to APO protein. All the ligands accepted Lipinski's rule of five. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis revealed only ED21 and ED5 as promising druglike candidates that can have high therapeutic value and no side effects. These in-silico findings contribute to the development of potent SARS-CoV-2 inhibitors, potentially advancing the quest for effective antiviral drugs.

Discovery of new thymol-3,4-disubstituted thiazole hybrids as dual COX-2/5-LOX inhibitors with in vivo proof

New thymol–3,4-disubstitutedthiazole hybrids were synthesised as dual COX-2/5-LOX inhibitors. Compounds 6b, 6d, 6e, and 6f displayed in vitro inhibitory activity against COX-2 (IC50= 0.037, 0.042, 0.046, and 0.039 µM) nearly equal to celecoxib (IC50= 0.045 µM). 6b, 6d, and 6f showed SI (379, 341, and 374, respectively) higher than that of celecoxib (327). 6a–l elicited in vitro 5-LOX inhibitory activity higher than quercetin. 6a–f, 6i–l, 7a, and 7c possessed in vivo inhibition of formalin induced paw edoema higher than celecoxib. 6a, 6b, 6f, 6h–l, and 7b showed gastrointestinal safety profile as celecoxib and diclofenac sodium in the population of fasted rats. Induced fit docking and molecular dynamics simulation predicted good fitting of 6b and 6f without changing the packing and globularity of the apo protein. In conclusion, 6b and 6f achieved the target goal as multitarget inhibitors of inflammation.

Comparative molecular dynamics simulation of apo and holo forms of the P53 mutant C176F: a structural perspective

Zinc fingers represent a highly diverse structural domain, with P53 being a notable example among zinc-dependent transcription factors. The folding patterns of proteins in the cell are heavily influenced by the concentration of zinc. The potential for zinc loss arises due to its dysregulation and the frequent occurrence of tumorigenic P53 mutations. This could lead to significant consequences such as protein misfolding and a reduction in tumor-suppressing capabilities. To gain deeper insights into the structural conformation, stability, flexibility, and compactness of the zinc-binding mutation C176F, a comprehensive comparative computational analysis was conducted on the wildtype (WT) and mutant (MT) P53 in the presence (Holo) and absence (Apo) of zinc. This analysis was performed using molecular dynamic simulation. The overall observation was that the mutation in C176F reduces the metal binding affinity and results in less stability in the Apo and Holo P53 MT protein.

Role of plasma LPL and GPIHBP1 in patients with pancreatitis

Plasma LPL and GPIHBP1 levels and other lipid metabolic factors in patients with pancreatitis with blood triglycerides in the normal range were measured over time. The subjects included patients with acute pancreatitis (AP) (n=57) and chronic pancreatitis (CP) (n=30), and 20 healthy controls. Plasma LPL and GPIHBP1 were measured by enzyme-linked immunosorbent assay, apo protein by immunoturbidimetric assay, and other lipids by routine methods. Disease status was based on the APACHE-II score at the time of blood sampling. AP cases showed a significant decrease in LPL and GPIHBP1 compared to controls, whereas CP cases showed a non-significant decrease. ApoC2 II and III levels in patients with pancreatitis were lower than those in controls. LPL and GPIHBP1 levels were significantly positively correlated with each other, and both showed a significant negative correlation with the APACHE-II score. LPL and GPIHBP1 levels in pancreatitis survivors and non-survivors showed a trend towards a decrease with worsening disease and an increase with recovery. Serum triglyceride levels showed a non-significant negative correlation with LPL and GPIHBP1 levels. These results suggest that pancreatitis is related to abnormal lipid metabolism, mainly through a decrease in LPL in the course of the disease.

Discovery and preclinical development of LY3473329, a potent small molecule inhibitor of lipoprotein(a) formation

Abstract Background Lipoprotein(a) [Lp(a)] is a lipoprotein particle consisting of a low-density lipoprotein (LDL) particle and apolipoprotein(a) [apo(a)]. Lp(a) is a causal risk factor for cardiovascular disease. The formation of Lp(a) requires an initial interaction of apo(a) Kringle IV Domains 7 and 8 (KIV7, KIV8) with apoB-100 on LDL, followed by formation of a disulfide bond between apo(a) and apoB-100. Blocking the initial apo(a)-apoB interaction is a viable therapeutic approach to reduce circulating levels of Lp(a). The apo(a) protein is encoded by the LPA gene that arose from the plasminogen gene through an evolutionary gene duplication event. Because of sequence conservation between apo(a) and plasminogen, compound selectivity for apo(a) is imperative for clinical development. Purpose We report the discovery and preclinical characterization of LY3473329, a novel selective small molecule inhibitor of Lp(a) formation. Methods Computational and synthetic chemistry approaches coupled with multiple biochemical and biophysical assays were used to create apo(a) KIV7,8 binders. Chemical optimization led to molecules with potent inhibition of in vitro Lp(a) formation. Both apo(a) and plasminogen binding assays were conducted to evaluate compound selectivity. The potential for LY3473329 to lower steady state Lp(a) levels was assessed in human Lp(a) transgenic mice and in cynomolgus monkeys, and its effect on plasminogen activity was assessed in rat and monkey. Results Computational modeling, synthetic chemistry and small molecule library screens identified compounds that bind apo(a) KIV7 and KIV8 domains, and subsequent chemical optimization yielded compounds with sub-micromolar Lp(a) inhibition potency. Exploration of compound multivalency created inhibitors with sub-nanomolar potency, including LY3473329. LY3473329 engages multiple apo(a) KIV motifs, allowing it to potently inhibit Lp(a) formation in vitro. Oral doses of LY3473329 given to human Lp(a) transgenic mice and cynomolgus monkeys caused dose-dependent decreases in plasma Lp(a). The key binding motif for LY3473329 is shared by two KIV domains in rat and monkey plasminogen, but only one KIV domain in human plasminogen. LY3473329 was non-selective for rat and monkey plasminogen, but showed ∼ 50-fold selectivity for human plasminogen in in vitro binding assays. In cynomolgus monkeys and rats, LY3473329 significantly reduced plasminogen activity levels without adverse effects. Given its selectivity against human plasminogen, LY3473329 is expected to lower Lp(a) in human without affecting plasminogen. LY3473329 has been evaluated in a human Phase 1 study and is currently in Phase 2 clinical development. Conclusions LY3473329 is a potent and selective small molecule inhibitor of Lp(a) formation and is the only orally administered Lp(a) investigational therapeutic in clinical development.

Gene expression analysis reveals GRIN1, SYT1, and SYN2 as significant therapeutic targets and drug repurposing reveals lorazepam and lorediplon as potent inhibitors to manage Alzheimer’s disease

Alzheimer’s disease (AD) is a slowly progressive neurodegenerative disease and a leading cause of dementia. We aim to identify key genes for the development of therapeutic targets and biomarkers for potential treatments for AD. Meta-analysis was performed on six microarray datasets and identified the differentially expressed genes between healthy and Alzheimer’s disease samples. Thereafter, we filtered out the common genes which were present in at least four microarray datasets for downstream analysis. We have constructed a gene-gene network for the common genes and identified six hub genes. Furthermore, we investigated the regulatory mechanisms of these hub genes by analysing their interaction with miRNAs and transcription factors. The gene ontology analysis results highlighted the enriched terms significantly associated with hub genes. Through an extensive literature survey, we found that three of the hub genes including GRIN1, SYN2, and SYT1 were critically involved in disease development. To leverage existing drugs for potential repurposing, we predicted drug-gene interaction using the drug-gene interaction database, and performed molecular docking studies. The docking results revealed that the drug compounds had strong interactions and favorable binding with selected hub genes. Lorazepam exhibits a binding energy of −7.3 kcal/mol with GRIN1, Lorediplon exhibits binding energies of −7.7 kcal/mol and −6.3 kcal/mol with the SYT1, and SYN2 respectively. In addition, 100 ns molecular dynamics simulations were carried out for the top complexes and apo protein as well. Furthermore, the MM-PBSA free energy calculations also revealed that these complexes are stable and had favorable energies. According to our study, the identified hub gene could serve as a biomarker as well as a therapeutic target for AD, and the proposed repurposed drug molecules appear to have promising efficacy in treating the disease. Communicated by Ramaswamy H. Sarma

Identifying Metal Binding Sites in Proteins Using Homologous Structures, the MADE Approach.

In order to identify the locations of metal ions in the binding sites of proteins, we have developed a method named the MADE (MAcromolecular DEnsity and Structure Analysis) approach. The MADE approach represents an evolution of our previous toolset, the ProBiS H2O (MD) methodology, for the identification of conserved water molecules. Our method uses experimental structures of proteins homologous to a query, which are subsequently superimposed upon it. Areas with a particular species present in a similar location among many homologous protein structures are identified using a clustering algorithm. Dense clusters likely represent positions containing species important to the query protein structure or function. We analyze well-characterized apo protein structures and show that the MADE approach can identify clusters corresponding to the expected positions of metal ions in their binding sites. The greatest advantage of our method lies in its generality. It can in principle be applied to any species found in protein records; it is not only limited to metal ions. We additionally demonstrate that the MADE approach can be successfully applied to predict the location of cofactors in computer-modeled structures, e.g., via AlphaFold. We also conduct a careful protein superposition method comparison and find our methodology robust and the results largely independent of the selected protein superposition algorithm. We postulate that with increasing structural data availability, additional applications of the MADE approach will be possible such as non-protein systems, water network identification, protein binding site elaboration, and analysis of binding events, all in a dynamic manner. We have implemented the MADE approach as a plugin for the PyMOL molecular visualization tool. The MADE plugin is available free of charge at https://gitlab.com/Jukic/made_software.

Enhancing Hit Discovery in Virtual Screening through Absolute Protein-Ligand Binding Free-Energy Calculations.

In the hit identification stage of drug discovery, a diverse chemical space needs to be explored to identify initial hits. Contrary to empirical scoring functions, absolute protein-ligand binding free-energy perturbation (ABFEP) provides a theoretically more rigorous and accurate description of protein-ligand binding thermodynamics and could, in principle, greatly improve the hit rates in virtual screening. In this work, we describe an implementation of an accurate and reliable ABFEP method in FEP+. We validated the ABFEP method on eight congeneric compound series binding to eight protein receptors including both neutral and charged ligands. For ligands with net charges, the alchemical ion approach is adopted to avoid artifacts in electrostatic potential energy calculations. The calculated binding free energies correlate with experimental results with a weighted average of R2 = 0.55 for the entire dataset. We also observe an overall root-mean-square error (RMSE) of 1.1 kcal/mol after shifting the zero-point of the simulation data to match the average experimental values. Through ABFEP calculations using apo versus holo protein structures, we demonstrated that the protein conformational and protonation state changes between the apo and holo proteins are the main physical factors contributing to the protein reorganization free energy manifested by the overestimation of raw ABFEP calculated binding free energies using the holo structures of the proteins. Furthermore, we performed ABFEP calculations in three virtual screening applications for hit enrichment. ABFEP greatly improves the hit rates as compared to docking scores or other methods like metadynamics. The good performance of ABFEP in rank ordering compounds demonstrated in this work confirms it as a useful tool to improve the hit rates in virtual screening, thus facilitating hit discovery.

Novel pyrimidines as COX-2 selective inhibitors: synthesis, DFT analysis, molecular docking and dynamic simulation studies

Pyrimidine and its derivatives are associated with varieties of biological properties. Therefore, we herein reported the synthesis of four novel pyrimidines (2, 3, and 4a, b) derivatives. The structure of these molecules is confirmed by spectroscopic methods such as IR, NMR, and Mass analysis. The electronic behavior of synthesized compounds 4a, b and in silico drug design 4 c, d was explained by Density Functional Theory estimations at the DFT/B3LYP level via 6-31 G++ (d, p) replicates the structure and geometry. All the synthesized compounds were screened for their in vitro COX-1 and COX-2 inhibitory activity compared to standards Celecoxib and Ibuprofen. Compounds 3 and 4a afforded excellent COX-1 and COX-2 inhibitory activities at IC50 = 5.50 and 5.05 μM against COX-1, 0.85 and 0.65 μM against COX-2, respectively. The standard drugs Celecoxib and Ibuprofen showed inhibitory activity at IC50 = 6.34 and 3.1 μM against COX-1, 0.56 and 1.2 μM against COX-2, respectively. Further, these compounds showed high potential docking with SARS-CoV-2 Omicron protease & COX-2 and predicted drug-likeness for the pyrimidine analogs by using Molinspiration. The protein stability, fluctuations of APO–protein, protein–ligand complexes were investigated through Molecular Dynamics simulations studies using Desmond Maestro 11.3 and potential lead molecules were identified. Communicated by Ramaswamy H. Sarma

Identification of Potential Lead Compounds Targeting Novel Druggable Cavity of SARS-CoV-2 Spike Trimer by Molecular Dynamics Simulations.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an urgent public health problem. Spike (S) protein mediates the fusion between the virus and the host cell membranes, consequently emerging as an important target of drug design. The lack of comparisons of in situ full-length S homotrimer structures in different states hinders understanding the structures and revealing the function, thereby limiting the discovery and development of therapeutic agents. Here, the steady-state structures of the in situ full-length S trimer in closed and open states (Sclosed and Sopen) were modeled with the constraints of density maps, associated with the analysis of the dynamic structural differences. Subsequently, we identified various regions with structure and property differences as potential binding pockets for ligands that promote the formation of inactive trimeric protein complexes. By using virtual screening strategy and a newly defined druggable cavity, five ligands were screened with potential bioactivities. Then molecular dynamic (MD) simulations were performed on apo protein structures and ligand bound complexes to reveal the conformational changes upon ligand binding. Our simulation results revealed that sulforaphane (SFN), which has the best binding affinity, could inhibit the conformational changes of S homotrimer that would occur during the viral membrane fusion. Our results could aid in the understanding of the regulation mechanism of S trimer aggregation and the structure-activity relationship, facilitating the development of potential antiviral agents.

Inactivation mechanism of an immunoglobulin modulator of the T-cell potassium channel Kv1.3.

Kv1.3 belongs to the Shaker family of voltage-activated potassium channels that are abundantly expressed in the T lymphocytes, where it plays an important role in T-cell activation. This role has made Kv1.3 a target for therapeutic immunomodulation for autoimmune diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Here, through cryo-electron microscopy (cryo-EM) and electrophysiology, we have investigated the modulatory mechanism of an inhibitory nanobody A0194009G09 (Ablynx/Sanofi). Functional studies reveal that the nanobody inhibits Kv1.3 by promoting slow C-type inactivation and that multiple nanobodies can bind to individual Kv1.3 channels. The structure of the human Kv1.3 in apo state (without modulators) showed a dynamic selectivity filter (SF) with two conformations (D1, D2) that are dilated when compared to reference structures where the SF is conducting. In the presence of the nanobody, the structure displays a SF in the D1 conformation, similar to the apo protein, but also a distinct conformation where the SF was further dilated (D3). Further, the nanobody-bound structure reveals how the nanobody bridges the turret loop within the pore domain and the S1-S2 loop within the voltage sensing domains to promote C-type inactivation. To explore how the nanobody promotes inactivation, we mutated residues at the interface between the nanobody and channel in both the S1-S2 loop and the turret loop, as well as a residue in the SF (H451) known to control inactivation in Kv1.3. Interestingly, the mutations that prevent inactivation in Kv1.3 largely attenuated the inhibitory action of the nanobody, similar to the mutations at the Kv1.3-nanobody binding interface. Taken together, our results demonstrate that the nanobody inhibits Kv1.3 by promoting inactivation, and we are currently solving mutant structures to elucidate the molecular mechanisms of how nanobody binding promotes inactivation.

Gold nanoparticle-coated apoferritin conductive nanowires.

Gold-metallic nanofibrils were prepared from three different iso-apoferritin (APO) proteins with different Light/Heavy (L/H) subunit ratios (from 0% up to 100% L-subunits). We show that APO protein fibrils have the ability to in situ nucleate and grow gold nanoparticles (AuNPs) simultaneously assembled on opposite strands of the fibrils, forming hybrid inorganic-organic metallic nanowires. The AuNPs are arranged following the pitch of the helical APO protein fiber. The mean size of the AuNPs was similar in the three different APO protein fibrils studied in this work. The AuNPs retained their optical properties in these hybrid systems. Conductivity measurements showed ohmic behavior like that of a continuous metallic structure.

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

Iron–sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe–S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe–4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron–sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered “SufFeScient” derivative of BL21(DE3). Our results show that co-expression of a Fe–S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe–S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe–S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe–S cluster maturation and points out the pitfalls of chemical reconstitution.Graphical abstract

A beginner’s guide to molecular docking

In this opinion, the basics of molecular docking (MD) such as binding affinity, binding pose, and ligand interactions with common docking-related terminologies (Apo protein, positive control, native ligand, co-crystal inhibitors) are discussed. We have provided different figures to aid in the graphical interpretation of the discussed literature. Following this, a few advantages (simplicity, fast, applicability) and disadvantages of MD are highlighted. This opinion will benefit bachelor and master students (or anyone) that are interested in learning the technique of MD. We encourage the sensible use of the MD technique and strict analysis to avoid interpretation errors in the results. The binding affinity, binding pose, and ligand interactions should be collectively considered during the result analysis. For every study, we strongly recommend a strict validation of the docking protocols.

Abstract 13135: Mir455 Targets the Repeated Domains of Lpa Open Reading Frame and Lowers Its Expression and The Secretion of Apolipoprotein (a) From Human Cells

Introduction: Lipoprotein (a) [Lp(a)] is a peculiar lipoprotein species containing a unique glycoprotein apolipoprotein(a) [apo(a)] encoded by the LPA gene. Lp(a) is causatively, independently and significantly associated with CAD and calcified aortic valve stenosis. MiRs are approximately 22 nucleotides in length and work by inducing RNA silencing, thus reducing target genes expression. The influence of these regulatory elements on the expression of LPA remains unknown. Hypothesis: Apo(a) and Lp(a) plasma levels are genetically regulated by miRs. Method: A prediction software was used to identify miR candidates targeting the human LPA mRNA open reading frame (ORF). We investigated the selected candidates by transfecting these miRs (i) in HEK293 cells stably expressing a short LPA isoform (i.e. 17 KIV domains) and (ii) in the Hep3B human hepatoma cell line that endogenously express LPA . A validated siRNA targeting LPA and a scrambled siRNA were used as positive and negative controls, respectively. LPA expression was assessed by RT-qPCR. Apo(a) protein expression and secretion in the culture medium were assessed by Western Blot and ELISA, respectively. Results: We identified four miR candidates (miR22, miR194, miR218, and miR455) based on sequence complementarity between their seed region and the LPA mRNA ORF. Compared with the negative control, only miR455 significantly reduced LPA mRNA expression (-67±8%) as well as apo(a) intracellular abundance (-41±9%) and apo(a) secretion (-36±9%) in the culture medium ( p<0.001 , all). Similar reductions were observed with the positive siRNA control (-77±4%, -41±11%, -53±11 %, p<0.001 all, respectively). No significant effect on LPA expression was seen with the three other miR candidates. Conclusion: These in vitro results suggest that at least one human miR has the potential to regulate the circulating levels of Lp(a) in vivo . This will be verified by measuring miR455 abundance in liver and plasma samples from individuals with high vs. low Lp(a) plasma concentrations. Our study also underlines that targeting LPA with interfering RNAs is likely a regulatory process occurring in humans.