Interdomain Region Research Articles

IgG AND CD4+ Responses Post Immunization With Cidr1α-Pfemp1 Recombinant Protein

Malaria vaccine is urgently needed to eradicate malaria. The complexity of Plasmodium sp life cycle and host immunity to malaria have become the forces for developing peptide-based malaria vaccines using recombinant technology. The CIDR1α-PfEMP1 (cysteine-rich interdomain region 1α of Plasmodium falciparum erythrocyte membrane protein 1) is vital in malaria pathogenicity, making it a malaria vaccine candidate. This study investigated the IgG and CD4+ responses generated after the CIDR1α-PfEMP1 recombinant protein immunization in Wistar rats. The rats were divided into a control group, injected with 0.9% NaCl, and a treatment group, subcutaneously injected with 150 µg of purified CIDR1α-PfEMP1 recombinant protein. Injection was conducted thrice with a three-week interval. Blood samples were extracted every two weeks after immunization. IgG and CD4+ concentrations were measured using ELISA. Data were analyzed using the independent T-test and Mann-Whitney test based on the data distribution. IgG and CD4+ concentrations increased along with the injection frequency. Significant differences between the control and treatment groups were observed in IgG concentrations after all injections and CD4+ level only after secondary II immunization (p<0.05). In conclusion, the CIDR1α-PfEMP1 recombinant protein induces humoral and cellular immune responses through increased IgG and CD4+ concentrations, making it a potential malaria vaccine candidate.

A systems serology approach to identifying key antibody correlates of protection from cerebral malaria in Malawian children

BackgroundPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined.MethodsIn Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes.ResultsThe selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80–96% interquartile range) and included antibody to well-characterized DBLβ3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLβ3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8–38% interquartile range) compared to cerebral malaria (7%, 30–15%, p < 0.001).ConclusionsAntibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.

Genetic evidence for a regulated cysteine protease catalytic triad in LegA7, a Legionella pneumophila protein that impinges on a stress response pathway.

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate MAPK activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on high osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain.

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

Recombinant Proteins Combining the Inter-domain Region of Pneumococcal Surface Antigen "A" and Adaptive W-type Polypeptide of Thermotoga Bacteria as a Potential Component Base for the Development of New Diagnostics and Genetically Engineered Subunit Vaccines against Pneumococcal Infection

The problems related to the development of pneumococcal vaccines require combination of traditional solutions and alternative systems optimizing this procedure. Recombinant subunit vaccines have undeniable advantages over inactivated and live-attenuated vaccines: they induce cell-mediated and humoral immunologic responses effectively and with high specificity, but without the risks associated with authentic pathogen processing. However, subunit vaccines require specific adjuvants to enhance the immune response or special fusion partners to improve solubility, expression and optimize subsequent fine purification of the protein of interest. In the framework of this work, a structurally conserved region of the most immunogenic region of the vaccine-valuable surface antigen PspA of Streptococcus pneumoniae was chosen as a model protein, and the adaptive polypeptide CheW from the hyperthermophilic microorganism Thermotoga petrophila was used as a promising fusion protein. Appropriate expression plasmid vectors were designed in silico and constructed in vitro. Efficient E. coli producer strains were obtained and appropriate conditions for heterologous production of chimeric proteins were selected. The fusion partner from T. petrophila positively influenced the properties of the resulting constructs such as thermostability, solubility, and homogeneity. During this work, the optimal pH and temperature ranges of the created proteins were determined, and the principles of low-stage purification were elaborated. We obtained and characterized new proteins, which were not previously found in nature in a similar bioconfiguration. The results indicate that the biotechnologically valuable characteristics of the fusion protein were more expressed when the adaptive CheW protein was combined with the N-terminus of the PspA antigen.

Prostate-specific membrane antigen (PSMA) glycoforms in prostate cancer patients seminal plasma.

Prostate-specific membrane antigen (PSMA) is a USFood and Drug Administration-approved theranostic target for prostate cancer (PCa). Although PSMA is known to be glycosylated, the composition and functional roles of its N-linked glycoforms have not been fully characterized. PSMA was isolated from pooled seminal plasma from low-risk grade Groups 1 and 2 PCa patients. Intact glycopeptides were analyzed by mass spectrometry to identify site-specific glycoforms. We observed a rich distribution of PSMA glycoforms in seminal plasma from low and low-intermediate-risk PCa patients. Some interesting generalities can be drawn based on the predicted topology of PSMA on the plasma membrane. The glycoforms at ASN-459, ASN-476, and ASN-638 residues that are located at the basal domain facing the plasma membrane in cells, are predominantly high mannose glycans. ASN-76 which is located in the interdomain region adjacent to the apical domain of the protein shows a mixture of high mannose glycans and complex glycans, whereas ASN-121, ASN-195 and ASN-336 that are located and are exposed at the apical domain of the protein predominantly possess complex sialylated and fucosylated N-linked glycans. These highly accessible glycosites display the greatest diversity in isoforms across the patient samples. Our study provides novel qualitative insights into PSMA glycoforms that are present in the seminal fluid of PCa patients. The presence of a rich diversity of glycoforms in seminal plasmaprovides untapped potential for glycoprotein biomarker discovery and as a clinical sample for noninvasive diagnostics of male urological disorders and diseases including PCa. Specifically, our glycomics approach will be critical in uncovering PSMA glycoforms with utility in staging and risk stratification of PCa.

CNSC-33. MOLECULAR AND FUNCTIONAL DRIVERS OF ACTIVITY DEPENDENT GLIOBLASTOMA PROLIFERATION

Abstract Glioblastoma, the most common primary brain tumor in adults, is a major cause of neurological morbidity and mortality with no effective therapies. Its proliferation and invasion are regulated by direct and paracrine-mediated neuronal activity. In this study, we uncover molecular targets and protein-protein signaling driving activity dependent proliferation. To identify candidate molecular pathways, we used high-density electrode arrays in vivo to record human local field potentials. Single-cell RNA sequencing identified Netrin-G1 (NTNG1) and thrombospondin-1 (TSP1) elevation in glioblastoma clonal population that proliferates in response to the presence of neurons. The amino acid sequence of NTNG1 and the TSR1 domain of TSP1 were utilized to predict the 3D interaction surface of NTNG1 and TSP1. Compared to structure predictions of NTNG1 and its known binding partner NGL1, NTNG1 and TSP1 complex predictions showed agreement between machine learning predicted models and lower predicted aligned error in interdomain regions of the complex, suggesting higher confidence in the relative position of NTNG1-TSP1 as compared to NTNG1-NGL1. We performed 45 protein structure predictions and two molecular dynamics simulations of mutant and WT TSP1 in complex with NTNG1. Each simulation box consisted of &amp;gt;263,000 atoms, each run for 50 million 2 femtosecond timesteps for a total simulation time of 200 ns. These calculations confirmed a stable complex in the WT condition and significant conformational change in the R46A condition, emphasizing the importance of this residue in mediating NTNG1-TSP1 binding. Computational affinity models between NTNG1 and TSP1 were validated by pull-down assays and immunofluorescence labelling using cerebral organoid and human glioma-neuronal coculture models. Finally, we design small molecule and fusion-protein inhibitors disrupting this interaction. Here we advance the development of targeted precision-medicine therapies to treat glioblastoma proliferation through activity-dependent mechanisms.

Identification of the flavivirus conserved residues in the envelope protein hinge region for the rational design of a candidate West Nile live-attenuated vaccine

The flavivirus envelope protein is a class II fusion protein that drives flavivirus-cell membrane fusion. The membrane fusion process is triggered by the conformational change of the E protein from dimer in the virion to trimer, which involves the rearrangement of three domains, EDI, EDII, and EDIII. The movement between EDI and EDII initiates the formation of the E protein trimer. The EDI-EDII hinge region utilizes four motifs to exert the hinge effect at the interdomain region and is crucial for the membrane fusion activity of the E protein. Using West Nile virus (WNV) NY99 strain derived from an infectious clone, we investigated the role of eight flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region in the conformational change of E protein from dimer to trimer and viral entry. Single mutations of the E-A54, E-I130, E-I135, E-I196, and E-Y201 residues affected infectivity. Importantly, the E-A54I and E-Y201P mutations fully attenuated the mouse neuroinvasive phenotype of WNV. The results suggest that multiple flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region play a critical role in the structure–function of the E protein and some contribute to the virulence phenotype of flaviviruses as demonstrated by the attenuation of the mouse neuroinvasive phenotype of WNV. Thus, as a proof of concept, residues in the EDI-EDII hinge region are proposed targets to engineer attenuating mutations for inclusion in the rational design of candidate live-attenuated flavivirus vaccines.

Dengue virus serotypic replacement of NS3 protease or helicase domain causes chimeric viral attenuation but can be recovered by a compensated mutation at helicase domain or NS2B, respectively.

Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%-78% amino acid identities, resulting in incomplete immunity, where one serotype's infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5' RNA triphosphatase (5'RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations' frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells. IMPORTANCE Enzyme activities for replicating DENV 5' cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5' cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)-NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.

Inter-domain interaction of ferredoxin-NADP+ reductase important for the negative cooperativity by ferredoxin and NADP(H).

Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH. The affinity between FNR and Fd is weakened by the allosteric binding of NADP(H) on FNR, which is considered as a part of negative cooperativity. We have been investigating the molecular mechanism of this phenomenon and proposed that the NADP(H)-binding signal is transferred to the Fd-binding region across the two domains of FNR, NADP(H)-binding domain and FAD-binding domain. In this study, we analyzed the effect of altering the inter-domain interaction of FNR on the negative cooperativity. Four site-directed FNR mutants at the inter-domain region were prepared, and their NADPH-dependent changes in the Km for Fd and physical binding ability to Fd were investigated. Two mutants, in which an inter-domain hydrogen bond was changed to a disulfide bond (FNR D52C/S208C) and an inter-domain salt bridge was lost (FNR D104N), were shown to suppress the negative cooperativity by using kinetic analysis and Fd-affinity chromatography. These results showed that the inter-domain interaction of FNR is important for the negative cooperativity, suggesting that the allosteric NADP(H)-binding signal is transferred to Fd-binging region by conformational changes involving inter-domain interactions of FNR.

Structural Insights into Phosphorylation-Mediated Polymerase Function Loss for DNA Polymerase β Bound to Gapped DNA.

DNA polymerase β is a member of the X-family of DNA polymerases, playing a critical role in the base excision repair (BER) pathway in mammalian cells by implementing the nucleotide gap-filling step. In vitro phosphorylation of DNA polymerase β with PKC on S44 causes loss in the enzyme's DNA polymerase activity but not single-strand DNA binding. Although these studies have shown that single-stranded DNA binding is not affected by phosphorylation, the structural basis behind the mechanism underlying phosphorylation-induced activity loss remains poorly understood. Previous modeling studies suggested phosphorylation of S44 was sufficient to induce structural changes that impact the enzyme's polymerase function. However, the S44 phosphorylated-enzyme/DNA complex has not been modeled so far. To address this knowledge gap, we conducted atomistic molecular dynamics simulations of pol β complexed with gapped DNA. Our simulations, which used explicit solvent and lasted for microseconds, revealed that phosphorylation at the S44 site, in the presence of Mg ions, induced significant conformational changes in the enzyme. Specifically, these changes led to the transformation of the enzyme from a closed to an open structure. Additionally, our simulations identified phosphorylation-induced allosteric coupling between the inter-domain region, suggesting the existence of a putative allosteric site. Taken together, our results provide a mechanistic understanding of the conformational transition observed due to phosphorylation in DNA polymerase β interactions with gapped DNA. Our simulations shed light on the mechanisms of phosphorylation-induced activity loss in DNA polymerase β and reveal potential targets for the development of novel therapeutics aimed at mitigating the effects of this post-translational modification.

A multiepitope chimeric antigen from Rhipicephalus microplus-secreted salivary proteins elicits anti-tick protective antibodies in rabbit

Rhipicephalus (Boophilus) microplus, the Cattle Fever Tick, causes significant economic losses in livestock in tropical and subtropical regions of the world. As the usual control strategy based on chemical acaricides presents different drawbacks, alternative control strategies have been considered for tick control. In recent decades, several tick proteins have been evaluated as targets for the development of anti-tick vaccines. Thus, in the present work, coding sequences from three different proteins present in tick saliva were employed together to construct a recombinant chimeric protein that was evaluated as an antigen in rabbit immunization. Then, the elicited antibodies were tested in a tick artificial feeding experiment to verify the protective effect against the parasites. In addition to Rhipicephalus microplus subtilisin inhibitor 7 (RmSI-7), a serine protease inhibitor member of the TIL (Trypsin Inhibitory Like) family, an interdomain region from the Kunitz inhibitor BmTI-A, and a new cysteine-rich AMP-like microplusin, called RmSEI (previously identified as an elastase inhibitor), were selected to compose the chimeric protein. Anti-chimeric IgG antibodies were able to affect R. microplus female egg production after artificial feeding. Moreover, antibodies elicited in infested tick-resistant and tick-susceptible cattle recognized the recombinant chimera. Additionally, the functional characterization of recombinant RmSEI was performed and revealed antimicrobial activity against gram-positive bacteria. Moreover, the antimicrobial protein was also recognized by antibodies elicited in sera from cattle previously exposed to R. microplus bites. Together, these data suggest that the chimeric protein composed of three salivary antigens is suitable for anti-tick vaccine development.

Block copolymer interfaces investigated by means of NMR, atomic force microscopy and dielectric spectroscopy

Self-assembly of block copolymers (BC) is a key feature for bottom-up patterning that is applied in the fabrication of templates for nanostructured membranes, photonic nanodevices, or solar cells. The wide range of possible BC applications is because the dissimilar blocks can be appropriately selected to form distinct domains with controllable dimensions and functionalities. An important representative of these materials is polystyrene-b-poly(ethylene oxide) (PS-b-PEO) diblock copolymer. This copolymer is of considerable academic interest and therefore it is examined by a plethora of experimental techniques to elucidate its structure and molecular dynamics. Particularly worth investigating is the interphase, consisting of polymer chains located at the interface of crystallite-amorphous and inter-domain regions, which codetermines the properties of such a system. It is shown that below the melting point of the PEO, the majority of the amorphous PEO are occupying interfacial sites and are represented by the rigid amorphous fraction (RAF) which can be monitored by dielectric and nuclear magnetic resonance spectroscopies. Moreover, two independent NMR spin-diffusion experiments reveal the asymmetrical nature of PS-PEO interface indicating a significant fraction of the PEO phase under the influence of stiff, adjacent polystyrene. An analysis of complementary thermal calorimetry and X-ray scattering data confirms the presence of microphase-separated morphology as well as the semicrystalline nature of PEO component. Lamelar domain architecture was confirmed by atomic force microscopy data acquired in the case of thin copolymer film.

Optimization of CIDR1α-PfEMP1 Gene Transformation in Eschericia coli BL21(DE3) by Heat-shock Method: Stages of Malaria Vaccine Development

Malaria is still a world health problem, especially in developing countries. Data from the World Malaria Report 2020, malaria cases in the world in 2019 reached 229 million cases with a total death of 409,000 people. The highest mortality occurred in patients under five, which reached 272,000 people. The high mortality rate of severe malaria worldwide needs multiple approaches including the malaria vaccine. Cysteine-rich Interdomain Regions 1α-Plasmodium falciparum Erythrocyte Membrane Protein 1 (CIDR1α-PfEMP1) is a candidate for malaria vaccine due to its important role in malaria pathogenesis. The development of CIDR1α-PfEMP1-based malaria vaccine is conducted by DNA recombinant technology where transformation is a pivotal step. The study aimed to optimize the transformation of CIDR1α-PfEMP1 into Escherichia coli BL21(DE3) using heat shock method to find out the highest transformation efficiency. The variables were duration of heat shock in 30, 50, and 70 secs and plating volume of 100 µL and 200 µL. The results showed that the highest transformation efficiency was 1.9 x 102 CFU/µg, it was achieved by 50 secs heat shock and 200 µL plating volume. This was an optimum transformation condition that can be used further for cloning of CIDR1α-PfEMP1 gene to develop a malaria vaccine.

Structural and dynamical effects of phosphorylation on DNA polymerase β studied by molecular dynamics simulations.

DNA polymerase β (pol β) is a member of the X-family of DNA polymerases that catalyze the distributive addition of nucleoside triphosphates during base excision DNA repair. Previous studies have shown that the enzyme was phosphorylated in vitro with PKC at two serines (44 and 55), causing the loss of DNA polymerase activity but not DNA binding. Here, we report a comprehensive atomic resolution study of wild-type and phosphorylated DNA polymerase in presence of Mg ions using microsecond-long molecular dynamics (MD) simulations. The results show drastic conformational changes in the structure of DNA polymerase β due to S44 phosphorylation. Phosphorylation-induced conformational changes transform the enzyme from a closed to an open structure. The dynamic cross-correlation calculations show that phosphorylation enhances correlated motions between different domains. Centrality network analysis reveals that the S44 phosphorylation causes structural rearrangements and modulates the information pathway between the Lyase domain and base pair binding domain. Further analysis of our simulations reveals that a critical hydrogen bond (between S44 and E335) disruption and the formation of three additional salt bridges are potential drivers of these conformational changes. In addition, we found that two of these additional salt bridges form in the presence of Mg ions on the active sites of the enzyme. Notably, we have identified phosphorylated-induced allosteric coupling between the inter-domain regions. Collectively, the result provides a mechanistic understating of conformational transitions observed due to the phosphorylation and reveals a putative allosteric site.

Expression and in silico Analysis of CIDRα1 Recombinant Protein from Plasmodium Falciparum as a Malaria Subunit Vaccine Candidate

Malaria vaccination is an essential approach to combat malaria. One major protein studied for vaccine development is Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1). It contains several important domains for malaria pathogenesis. The binding of Cysteine-rich interdomain region α1 (CIDRα1) of PfEMP1 to endothelial protein C receptor (EPCR) is associated with cerebral malaria, while CIDRα1 binding to CD36 has been correlated with uncomplicated malaria. The vital function of CIDRα1 of PfEMP1 makes it a potential vaccine candidate to prevent clinical features of malaria. A long journey of vaccine development can be shortened by the advancement of bioinformatics and biotechnology techniques. This study aimed to express the recombinant CIDRα1 of PfEMP1 and investigate its potency as a malaria subunit vaccine candidate by in silico analysis. Constructed CIDRα1-PfEMP1 was expressed in E. coli BL21(DE3) after induction with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purified using Ni-NTA column. In silico analysis on CIDRα1 of PfEMP1 sequence was conducted using ProtParam Tool for its physicochemical properties, Iterative Threading ASSEmbly Refinement (I-TASSER) server and JPred4 program to predict secondary structure, 3D modelling, and ligand-binding site, BepiPred 2.0 and Kolaskar-Tangaonkar to predict B-cell epitope, NetCTL server to determine T-cell epitope, and Vaxijen v2.0 server to predict its antigenicity. The chimeric CIDRα1 of PfEMP1 protein had a 27 kDa molecular weight and was classified as a stable protein. The secondary structure consisted of 6 helices connected with loops. It revealed similarity to CD36-binding protein, EPCR-binding domain, and protein involved in rosetting. The 3D structure modelling demonstrated conserved ligand-binding sites and accessible surface area, which are vital for receptor binding. It had B-cell and T-cell epitopes and was non-allergenic. The properties of the chimeric CIDRα of PfEMP1 indicated its potential as a malaria subunit vaccine candidate. HIGHLIGHTS The binding capacity of CIDRα1 of PfEMP1 to endothelial protein C receptor (EPCR) and CD36 makes it a potential vaccine candidate to prevent clinical malaria The chimeric CIDRα1 of PfEMP1 protein was a stable protein and showed similarity to CD36-binding protein, EPCR-binding domain, and protein involved in rosetting, which demonstrated conserved ligand-binding sites and accessible surface area, which are vital for receptor binding The chimeric CIDRα1 of PfEMP1 protein had B-cell and T-cell epitopes and was non-allergenic in in silico analysis, indicating its potential as a malaria vaccine candidate GRAPHICAL ABSTRACT

Structure-Based Virtual Screening and De Novo Design to Identify Submicromolar Inhibitors of G2019S Mutant of Leucine-Rich Repeat Kinase 2.

Missense mutations of leucine-rich repeat kinase 2 (LRRK2), including the G2019S mutant, are responsible for the pathogenesis of Parkinson's disease. In this work, structure-based virtual screening of a large chemical library was carried out to identify a number of novel inhibitors of the G2019S mutant of LRRK2, the biochemical potencies of which ranged from the low micromolar to the submicromolar level. The discovery of these potent inhibitors was made possible due to the modification of the original protein-ligand binding energy function in order to include an accurate ligand dehydration energy term. The results of extensive molecular docking simulations indicated that the newly identified inhibitors were bound to the ATP-binding site of the G2019S mutant of LRRK2 through the multiple hydrogen bonds with backbone amide groups in the hinge region as well as the hydrophobic interactions with the nonpolar residues in the P-loop, hinge region, and interdomain region. Among 18 inhibitors derived from virtual screening, 4-(2-amino-5-phenylpyrimidin-4-yl)benzene-1,3-diol (Inhibitor 2) is most likely to serve as a new molecular scaffold to optimize the biochemical potency, because it revealed submicromolar inhibitory activity in spite of its low molecular weight (279.3 amu). Indeed, a highly potent inhibitor (Inhibitor 2n) of the G2019S mutant was derived via the structure-based de novo design using the structure of Inhibitor 2 as the molecular core. The biochemical potency of Inhibitor 2n surged to the nanomolar level due to the strengthening of hydrophobic interactions in the ATP-binding site, which were presumably caused by the substitutions of small nonpolar moieties. Due to the high biochemical potency against the G2019S mutant of LRRK2 and the putatively good physicochemical properties, Inhibitor 2n is anticipated to serve as a new lead compound for the discovery of antiparkinsonian medicines.

Leukocyte and IgM Responses to Immunization with the CIDR1α-PfEMP1 Recombinant Protein in the Wistar Rat.

The malaria vaccine is an important strategy for the global malaria elimination program, but the complexity of the Plasmodium antigen is a major hurdle in malaria vaccine development. The cysteine-rich interdomain region 1α (CIDR1α) of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is crucial in malaria pathogenesis, making it a vaccine candidate. This study investigated the leukocyte and IgM response generated after administering a CIDR1α-PfEMP1 recombinant protein injection in Wistar rats. The rats were divided into a control group, who received a physiological saline solution (PSS), and a treatment group, who were subcutaneously injected with 150 µg of purified CIDR1α-PfEMP1 protein three times at the 3-week interval. Blood samples were collected every week after each injection. The number of leukocytes were counted using a Neubauer chamber, and the IgM concentration was determined using an enzyme-linked immunosorbent assay (ELISA). Data were analyzed using an independent, paired-T test, a Mann–Whitney test, and a Wilcoxon test, based on the distribution of the data. The total number of leukocytes notably increased on day 29 (p < 0.05). The percentage of neutrophils decreased, especially on day 8 (p < 0.05), whereas the percentages of monocytes and lymphocytes increased, primarily on day 14 (p < 0.05). The IgM concentration increased on day 14 (p < 0.05). In conclusion, the CIDR1α-PfEMP1 recombinant protein may induce leukocyte and IgM responses, making it a potential malaria vaccine candidate.

Structural and Dynamic Effects of PTEN C-Terminal Tail Phosphorylation.

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene encodes a tightly regulated dual-specificity phosphatase that serves as the master regulator of PI3K/AKT/mTOR signaling. The carboxy-terminal tail (CTT) is key to regulation and harbors multiple phosphorylation sites (Ser/Thr residues 380-385). CTT phosphorylation suppresses the phosphatase activity by inducing a stable, closed conformation. However, little is known about the mechanisms of phosphorylation-induced CTT-deactivation dynamics. Using explicit solvent microsecond molecular dynamics simulations, we show that CTT phosphorylation leads to a partially collapsed conformation, which alters the secondary structure of PTEN and induces long-range conformational rearrangements that encompass the active site. The active site rearrangements prevent localization of PTEN to the membrane, precluding lipid phosphatase activity. Notably, we have identified phosphorylation-induced allosteric coupling between the interdomain region and a hydrophobic site neighboring the active site in the phosphatase domain. Collectively, the results provide a mechanistic understanding of CTT phosphorylation dynamics and reveal potential druggable allosteric sites in a previously believed clinically undruggable protein.

Optimized Purification of CIDRα-PfEMP1 Plasmodium falciparum Recombinant Protein with Affinity Chromatography

Interaction of Cysteine-rich Interdomain Region (CIDR)α-Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) and Endothelial Cell Receptors especially CD36 on host cells is main malaria pathogenesis, makes this domain as a malaria vaccine candidate. Recently, the development of the malaria vaccine is conducted by recombinant technology, and the purification of the CIDRα-PfEMP1 recombinant protein is a pivotal step. This study aimed to determine an optimal condition to purify the CIDRα-PfEMP1 recombinant protein by affinity chromatography through imidazole and NaCl concentration. The purified recombinant protein was visualized using SDS-PAGE and its concentration was measured using Image J software and Bradford Assay. The data were analyzed using SPSS 26 software, and the Paired T-Test analysis was conducted to compare the concentration of purified recombinant protein from two different methods. The result showed that thetarget band of purified recombinant protein was 27 kDa. The thickest target protein band was observed in purified recombinant protein using 140 mM imidazole and 300 mM NaCl. The recombinant protein concentration using Image J software was 0.025 µg/µL, while the Bradford Assay was 0.56 µg/µL. The Paired T-Test analysis has a significance value of 0.010 (p&lt;0.05), meaning there was a significant difference between the concentration measurement using Image J software and Bradford Assay. In conclusion, the optimized condition to purify the CIDRα-PfEMP1 recombinant protein by affinity chromatography was using 140 mM imidazole and 300 mM NaCl. It is suggested to measure the purified CIDRα-PfEMP1 recombinant protein concentration using the Bradford Assay method due to its convenience and sensitivity.