Substitution Of Glutamic Acid Research Articles

Substitution of Glutamic Acid at Position 71 of DRβ1*04:01 Induces Collagen-Specific Tolerance Without Inducing Alloreactivity.

The DRB1 locus is strongly associated with both susceptibility and resistance to rheumatoid arthritis (RA). DRB1 alleles encoding the VKA or VRA epitope in positions 11, 71 and 74 confer the highest risk of developing RA, while the allele encoding VEA is protective. We therefore investigated the feasibility of creating antigen-specific tolerance without inducing alloreactivity by replacing lysine with glutamic acid at position 71 in DRβ1*04:01. Individual DRB1 alleles and the DRB1*04:01K71E allele were cloned into T2 cell lines to measure binding of biotinylated peptides. Transgenic animals expressing DRB1*04:01, DRB1*01:01 or DRB1*04:01K71E were injected with collagen to measure T cell proliferation. Skin and bone marrow transplants between DRB1*04:01K71E and DRB1*04:01 mice were performed to determine if the single amino acid change at position 71 would be recognized as foreign. DRB1*04:01 mice transplanted with DRB1*04:01K71E bone marrow were injected with collagen to test if resistance to collagen sensitization could be transferred. Replacing lysine (K) at position 71 in DRβ1*04:01 with glutamic acid (E) blocked collagen peptide binding and rendered the DRB1*04:01K71E mice resistant to collagen sensitization. Skin and bone marrow transplants from DRB1*04:01K71E mice were not rejected by DRB1*04:01 mice, suggesting the single E71 difference was not recognized as allogeneic. Bone marrow from DRB1*04:01K71E mice adoptively transferred antigen-specific tolerance to collagen to DRB1*04:01 mice. These studies demonstrate that editing a single amino acid in DRβ1*04:01 blocks collagen peptide binding without inducing alloreactivity and could therefore represent a gene therapy approach to induce antigen-specific passive tolerance.

Two Novel Variants in the CHRNA2 and SCN2A Genes in Italian Patients with Febrile Seizures.

Febrile seizures (FSs) are the most common form of epilepsy in children aged between six months and five years. The exact cause is unknown, but several studies have demonstrated the importance of genetic predisposition, with increasing involvement of receptors and ion channels. The present study aims to identify novel pathogenic variants in Italian patients with FSs. We performed targeted panel sequencing in a cohort of 21 patients with FSs. In silico analysis was performed to predict the pathogenic role of the resulting variants. We found two novel variants segregating in two families with FSs: c.1021C>G (p.Leu341Val) in the CHRNA2 gene and c.140A>G (p.Glu47Gly) in SCN2A. The c.1021C>G (p.Leu341Val) variant leads to a codon change of highly conserved leucine to valine at position 341 and is located in segments M3 of the subunit, which is important for channel gating. The c.140A>G (p.Glu47Gly) variant causes a substitution of glutamic acid with glycine at position 47 of the protein, which is highly conserved across the species. Moreover, it is located in the N-terminal domain, a region commonly affected in ASD, which impacts the inactivation kinetics and voltage dependence of steady-state activation. Further analyses are needed to better explain the role of CHRNA2 and SCN2A in the development of febrile seizures.

Abstract Or101: Phospho-Mimic Mutation in Beta Tubulin Suppresses Arrhythmias and Improves Cardiac Connexin 43 Localization in Duchenne Muscular Dystrophy Mice

Duchenne Muscular Dystrophy (DMD) is a severe X-linked neuromuscular disorder, causing progressive muscle degeneration and premature death due to heart and skeletal muscle dysfunction. It is caused by mutations in the dmd gene, leading to the absence of dystrophin, a crucial protein for muscle integrity. There is no cure for DMD. Its associated dystrophic cardiomyopathy is the primary cause of mortality by age 20. Connexin-43 (Cx43) is a gap junction protein responsible for electrical signaling in the heart. Cx43 localizes at the intercalated disc of the cardiomyocyte (CM), forming an intercellular channel with adjacent CMs; In DMD, Cx43 hemichannel remodels to the lateral side of the cardiomyocyte. Our previous research indicated that phospho-mimic Cx43 carrying serine to glutamic acid substitutions at the triplet regulatory site S325, S328, S330 in mdx hearts suppressed Cx43 lateralization and improved cardiac rhythm. The phospho-mimic Cx43 also reduced pathological hyperdensity and transverse directionality of microtubules in mdx cardiomyocytes. Colchicine (Colch) treatment, known to inhibit microtubule polymerization, also improved cardiac rhythm and Cx43 localization in mdx mice. To phenocopy the effect observed with Colch, we generated a mouse model with a beta tubulin mutation at serine 172, which was replaced by glutamic acid (S172E). We found reduced arrhythmias and improved localization of Cx43 in mdx hearts carrying the S172E phospho-mimic mutant tubulin by electrocardiography and immunofluorescent staining, respectively. Moreover, the proteomics data showed that the level of phospho-Cx43 was also upregulated in mdx hearts with S172E tubulin mutation. Further investigation will explore how this mutation impacts protein regulation and electrophysiology, and elucidate its mechanism of action in microtubule polymerization. These studies investigate a regulatory link between microtubule dynamics and localization of Cx43, potentially suggesting a novel therapeutic target for DMD-associated cardiomyopathy.

A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, invitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Phospho-mimetic CD3ε variants prevent TCR and CAR signaling.

Antigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome. We mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM. Integrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK. The data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CARsignaling and highlights the key role of CD3ε signalosome to tune signal transduction.

Hospitalization Events Among Adolescents and Adults With Sickle Cell Disease in a Tertiary Care Center in Central India.

Sickle cell disease (SCD) is an inherited red blood cell disorder, wherein mutation causes the substitution of glutamic acid to valine at the sixth position of the β-globin chain. These include sickle cell anemia (homozygous sickle mutation), sickle-beta thalassemia, and hemoglobin SCD. The clinical manifestations of SCD are protean. Individuals with SCD suffer from both acute and chronic complications, which include recurring episodes of pain commonly called vaso-occlusive crisis (VOC) - acute chest syndrome (ACS); aseptic necrosis of the bone; micro-infarction of the spleen, brain, and kidney; infections; stroke; and organ damage affecting every part of the body. SCD necessitates frequent hospitalizations because of severe complications, which pose a significant burden on caregivers and economic strain on healthcare systems. The pattern of hospital admission with SCD varies in different parts of the world. This study aimed to determine the causes of hospitalization among adolescent and adult patients with SCD and to determine factors associated with their hospital stay. The study was a hospital-based prospective observational study comprising adolescent and adult patients diagnosed with SCD, aged 15-45 years, who were hospitalized in the Department of General Medicine at All India Institute of Medical Sciences in Raipur from August 2021 to August 2022. According to our study, the primary reason for hospitalization was a painful crisis, accounting for 63% of cases, followed by infection (17%), ACS (11%), and acute hemolytic crisis (9%). Notably, we did not observe any significant differences between genders and causes of admission (p > 0.05). Joint pain (p = 0.005), back pain (p = 0.001), and chest pain (p = 0.001) were more frequently reported by adults over the age of 19. In addition, our analysis of the duration of hospital stays and various factors revealed that patients admitted for infections had a significantly longer mean hospital stay duration (p = 0.040). Acute painful crises were the primary cause of hospital admission among individuals with SCD; many patients also encountered infections and ACS. Furthermore, patients who experienced infections and VOC had a lengthier duration of hospital stay. Therefore, it is essential to provide them with comprehensive instructions on various preventive measures against infections and the factors that trigger painful crises.

Allele mining for Granule Bound Starch Synthase1 (GBSS1) gene governing amylose content in aromatic rice (Oryza sativa L.) germplasm

The current research investigated sequence polymorphism and allelic variation in the GBSS1 gene responsible for determining the apparent amylose content (AAC) in an aromatic rice germplasm panel. Analysis of 271 germplasm accessions from India revealed a wide range of variation in AAC, from 4.81% (Manipur Black Rice) to 35.88% (IGSR -3-1-5). Re-sequencing and sequence analysis of a representative subset of 28 accessions unveiled a nucleotide diversity (π) of 0.00374, identifying 72 sequence variants. Two novel non-synonymous SNPs were identified: a G/A transition in exon 2, resulting in the substitution of glutamic acid at the 91st position with lysine, and a C/T transition in exon 11, substituting alanine at the 468thposition with valine. Additionally, two previously reported SNPs were validated: A/C in exon 6 (Tyr224Ser) and C/T in exon 10 (Pro415Ser). Furthermore, a 23 bp exon 2 duplication was found in Manipur Black Rice (4.81%), Bhogali Bora (6.20%), and NJ-72 (7.36%), indicating the prevalence of this allele in the very low AAC category (3–9%) in addition to the glutinous or waxy rice category with 0–2% AAC. These findings are significant for manipulating starch content in aromatic rice germplasm.

Bone Health Impairment in Patients with Hemoglobinopathies: From Biological Bases to New Possible Therapeutic Strategies.

Hemoglobinopathies are monogenic disorders affecting hemoglobin synthesis. Thalassemia and sickle cell disease (SCD) are considered the two major hemoglobinopathies. Thalassemia is a genetic disorder and one of the major hemoglobinopathies determined by an impairment of globin chain production, which causes an alteration of erythropoiesis, an improvement in hemolysis, and an alteration of iron homoeostasis. In SCD, the mutations are on the β-globin chain of hemoglobin which results in a substitution of glutamic acid by valine with consequent formation of Hemoglobin S (HbS). Several factors are involved in bone metabolism alteration in patients with hemoglobinopathies, among them hormonal deficiency, bone marrow hyperplasia, iron overload, inflammation, and increased bone turnover. Bone metabolism is the result of balance maintenance between bone deposition and bone resorption, by osteoblasts (OBs) and osteoclasts (OCs). An impairment of this balance is responsible for the onset of bone diseases, such as osteoporosis (OP). Therefore, here we will discuss the alteration of bone metabolism in patients with hemoglobinopathies and the possible therapeutic strategies to contain and/or counteract bone health impairment in these patients, taking into consideration not only the pharmacological treatments already used in the clinical armamentarium, but also the new possible therapeutic strategies.

A Review of the Relationship between the Immune Response, Inflammation, Oxidative Stress, and the Pathogenesis of Sickle Cell Anaemia.

Sickle cell anaemia (SCD) is a life-threatening haematological disorder which is predominant in sub-Saharan Africa and is triggered by a genetic mutation of the β-chain haemoglobin gene resulting in the substitution of glutamic acid with valine. This mutation leads to the production of an abnormal haemoglobin molecule called haemoglobin S (HbS). When deoxygenated, haemoglobin S (HbS) polymerises and results in a sickle-shaped red blood cell which is rigid and has a significantly shortened life span. Various reports have shown a strong link between oxidative stress, inflammation, the immune response, and the pathogenesis of sickle cell disease. The consequence of these processes leads to the development of vasculopathy (disease of the blood vessels) and several other complications. The role of the immune system, particularly the innate immune system, in the pathogenesis of SCD has become increasingly clear in recent years of research; however, little is known about the roles of the adaptive immune system in this disease. This review examines the interaction between the immune system, inflammation, oxidative stress, blood transfusion, and their effects on the pathogenesis of sickle cell anaemia.

Mechanisms involved in hereditary angioedema with normal C1-inhibitor activity.

Patients with the inherited disorder hereditary angioedema (HAE) suffer from episodes of soft tissue swelling due to excessive bradykinin production. In most cases, dysregulation of the plasma kallikrein-kinin system due to deficiency of plasma C1 inhibitor is the underlying cause. However, at least 10% of HAE patients have normal plasma C1 inhibitor activity levels, indicating their syndrome is the result of other causes. Two mutations in plasma protease zymogens that appear causative for HAE with normal C1 inhibitor activity have been identified in multiple families. Both appear to alter protease activity in a gain-of-function manner. Lysine or arginine substitutions for threonine 309 in factor XII introduces a new protease cleavage site that results in formation of a truncated factor XII protein (Δ-factor XII) that accelerates kallikrein-kinin system activity. A glutamic acid substitution for lysine 311 in the fibrinolytic protein plasminogen creates a consensus binding site for lysine/arginine side chains. The plasmin form of the variant plasminogen cleaves plasma kininogens to release bradykinin directly, bypassing the kallikrein-kinin system. Here we review work on the mechanisms of action of the FXII-Lys/Arg309 and Plasminogen-Glu311 variants, and discuss the clinical implications of these mechanisms.

Progress of CRISPR/Cas9 in the treatment of sickle cell disease

Sickle cell disease (SCD) is a hereditary monogenic disease, which is characterized by the substitution of glutamic acid at the sixth position of β-peptide chain by valine, resulting in insufficient hemoglobin synthesis and a serious threats to human health. SCD causes about 80% of mortality and morbidity worldwide. Patients with SCD who are homozygous usually die before the age of 30. Patients with SCD who are heterozygous usually have a better prognosis because of their low intraerythrocytic hemoglobin S. However, FDA-approved drugs such as hydroxyurea and L-glutamine can reduce the severity of the disease, and there is no definite cure for all patients with SCD. At this stage, many scholars use creation and correction methods to induce pluripotent stem cells (IPSCs) to treat sickle cell disease. Gene editing is currently considered to be one of the most potential methods for the treatment of SCD. Among them CRISPR/Cas9 gene editing engineering technology has already made an abyss contribution to the transformation of genome engineering and its application in clinical or medical experiments. This review systematically introduces the gene therapy of CRISPR/Cas9, and summarizes the challenges and prospects of this technique in the treatment of sickle cell anemia.

Design of supported organocatalysts from a biomass-derived difuran compound and catalytic assessment for lactose hydrolysis.

The engineered structures and active sites of enzyme catalysts give rise to high catalytic activity and selectivity toward desired reactions. We have employed a biomass-derived difuran compound to append N-substituted maleimides with amino acid (glutamic acid) substitution by Diels-Alder reaction to mimic the chemical functional groups that comprise the active site channels in enzyme catalysts. The difunctionality of the biomass-derived difuran allows production of Diels-Alder adducts by appending two amino acid moieties to form a difunctional organocatalyst. The catalytic activity of the organocatalyst can be improved by immobilizing the organocatalyst on solid supporting materials. Accordingly, the structures of these immobilized organocatalysts can be engineered to mimic enzymatic active sites and to control the interaction between reactants, products, and transition states of catalytic reactions. Lactose hydrolysis was carried out to provide an example of industrial application of this approach to design and fabricate new supported organocatalysts as artificial enzymes.

The Severity of Sickle Cell Anemia in Men - The Action of Molecular and Biochemical Markers

Sickle cell disease is characterized by a very heterogeneous clinical course among patients with the same mutations for sickle cell hemoglobin (HbS). Sickle cell anemia (SCA) is a hereditary hemoglobinopathy caused by the homozygosity of a point mutation in the beta-globin gene, which leads to the substitution of glutamic acid for valine in the sixth position.

P347: COOPERATION OF TLX3 AND FLT3-ITD IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IS ENHANCED BY TLE4 INACTIVATION

Background: One fifth of T-cell acute lymphoblastic leukemia (T-ALL) cases is defined by the aberrant expression of TLX3. Being similar to the closely related TLX1, its expression is mainly associated with mutations in the JAK/STAT pathway (Liu et al., Nat Genetics 2017), which have been shown to cooperate with TLX1 in driving T-ALL (Vanden Bempt et al., Cancer Cell 2018). However, it is not known whether TLX3 has the same oncogenic role as TLX1. Additionally, TLX3 expression is also regularly found with FLT3-ITD mutations, which are virtually absent in TLX1-positive cases (Liu et al., Nat Genetics 2017), suggesting there are some important functional differences between TLX3 and TLX1. Aims: To set up a model of cooperation between TLX3 and FLT3-ITD and to elucidate the oncogenic role of TLX3 in T-ALL. Methods: We performed in vivo bone marrow transplant (BMT) assays by transducing lineage-negative cells derived from C57BL/6 mice with the respective oncogenes using retroviruses. Pro-T cell growth curves were performed in the absence of interleukin-7 (Il7) as described previously (Van Thillo et al., Nat Communications 2021). Fluorescence activated cell sorting (FACS) staining was performed on the MACSQuant VYB (Miltenyi Biotec) or Fortessa (BD). RNA was prepared at the KU Leuven Genomics Core and 3’ prime end sequencing (QuantSeq) was performed. Differential gene expression analyses were conducted using the DeSeq2 R package (v1.22.0). Results: The combination of TLX3 and FLT3-ITD in a constitutive BMT assay resulted in a rapid leukemia with an immature myeloid immunophenotype (CD11b+, Gr1-, CD4-, CD8-). The leukemia cells were positive for both FLT3-ITD (GFP) and TLX3 (mCHERRY), indicating cooperation between the two oncogenes in vivo. Similarly, in our in vitro pro-T-cell model, the TLX3 and FLT3-ITD double-positive cells outcompeted the single-positive cells after withdrawal of Il7. Next, we performed RNA-sequencing in CreER (estrogen receptor) pro-T cells 24 hours after activation of TLX3 expression by adding tamoxifen. This showed both up- and downregulation of many genes, including Tle4 among the downregulated genes. Interestingly, TLE4 expression was also lowest in the TLX3-subgroup of patients (Liu. et al. Nat Genetics 2017). TLE proteins are transcriptional co-repressors that can interact with the engrailed-homology 1 (Eh1) domain of transcription factors (Riz et al. Biochem Biophys Res Commun 2009). Therefore, we inserted a point mutation in the Eh1 domain of TLX3, resulting in a phenylalanine to glutamic acid substitution at position 18, to impair this interaction. Strikingly, TLX3(F18E) in combination with FLT3-ITD led to a proliferative advantage in pro-T cells after the omission of Il7 compared to wild-type TLX3. On top of that, cells with TLX3(F18E) and FLT3-ITD were able to grow in the absence of stem cell factor (Scf). Summary/Conclusion: We demonstrate that TLX3 cooperates with FLT3-ITD in vivo and in vitro in a pro-T-cell context. TLX3 downregulates the co-repressor Tle4 and inhibiting the interaction between TLX3 and Tle4 reinforces the proliferative capacity of pro-T cells in the absence of growth factors.

Liver gene therapy: The magic bullet for the sick lung

Alpha1-antitrypsin (AAT) deficiency is one of the most common genetic diseases and is due to pathogenic variants in the SERPINA1 gene that encodes for AAT, an acute-phase protein with anti-inflammatory and immunomodulatory properties. Although more than 150 pathogenic variants have been described, 95% of individuals with severe AAT deficiency are homozygotes for the Z allele, a substitution of glutamic acid for lysine at residue 342 (p.Glu342Lys) in SERPINA1. This single amino acid change makes Z-AAT prone to aggregation mostly in hepatocytes, the cells that abundantly synthetize and secrete AAT into the circulation.

An interesting case of anemia with jaundice

Hemoglobinopathies are haematalogical disorders that afflict millions of individuals worldwide. HbE is a hemoglobin variation caused by a mutation in the β globin gene that results in the substitution of glutamic acid for lysine at position 26 of the β globin gene. Hemoglobin (Hb) synthesis abnormalities are among the most prevalent inherited disorders. They can be quantitative (thalassemia syndrome) or qualitative (variant HbS). Hemoglobin E (HbE) is the second most common hemoglobin variation after hemoglobin S (HbS).

Role of the conserved E2 residue G259 in classical swine fever virus production and replication

The E2 glycoprotein of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle. The chimeric live attenuated C strain with the E2 substitution of bovine viral diarrhea virus (BVDV) is a promising marker vaccine candidate. In this study, the recombinant chimeric CSFV/bE2 cDNA clone harboring heterologous E2 (bE2) of BVDV was constructed by genetic approaches. Recombinant infectious virus rCSFV/bE2 (P11) was recovered by 11 serial passages of transfected PK15 cells. Viral genome sequencing showed that a glutamic acid to glycine mutation (E260G) at position 260 of the bE2 was observed in rCSFV/bE2 P11. Alignment of amino acid sequences displayed that the glycine was one of three conserved residues in pestivirus E2. When the glutamic acid to glycine substitution (E260G) was introduced into chimeric CSFV/bE2 cDNA clone, the high-titer infectious rCSFV/bE2E260G was rescued. The glycine to glutamic acid substitution at corresponding position in CSFV E2 resulted in significantly decreased rCSFV/E2G259E production. We further identified that the conserved E2 residue G259 played a critical role in the release and binding activity of CSFV and that the E2 residues G259 and V111 modulated synergistically infectious virus production and replication.

A mechanism for hereditary angioedema caused by a lysine 311–to–glutamic acid substitution in plasminogen

AbstractPatients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Single nucleotide polymorphism genotyping of ALDH2 gene based on asymmetric PCR and fluorescent probe-mediated melting curves

Detection of single nucleotide polymorphisms (SNPs) is of great value in precision medicine. The polymorphism of the aldehyde dehydrogenase 2 (ALDH2) gene is caused by a G1510A transition, resulting in the substitution of glutamic acid by lysine at position 487. People of different ALDH2 genotypes show different susceptibility to cancer, metabolic diseases, etc. SNP analysis based on fluorescent probe-mediated melting curves is a relatively efficient and cost-effective method. Genomic DNA extracted from 100 whole blood samples was subjected to polymorphisms mutational analysis using asymmetric PCR and probe-mediated melting curves. Then a certain number of samples from each genotype were randomly selected for direct sequencing verification. The new assay can be performed in 2 h without post-PCR processing such as gel electrophoresis and validated by direct sequencing in a blind study with 100% concordance. Moreover, comparing the detection of polymorphisms of ALDH2 with the clinics, and an overall agreement of 100% (100/100) was demonstrated. Our study has shown a high level of concordance between DNA sequencing, which is suitable for the detection of clinical specimens. Based on the concept of probe-mediated melting curves, we further developed this platform as a universal strategy for the detection of polymorphisms related to folate metabolism.

Abstract P061: De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics

Abstract Background: Research focused on the application of nuclear localization sequence (NLS)-based therapeutics has been a topic of intense interest to medicine since the core principles governing nuclear transport were awarded the Nobel Prize in 1999. Despite these efforts, efficient nuclear localization has been difficult. A major obstacle is that NLSs have cationic-net charges (abundance of lysines/arginines) required for appropriate interactions with the nuclear transporter importin-a (Impa). Once in the blood stream, cationic charges can cause strong non-specific cellular uptake and are rapidly cleared from the plasma compartment, which prevents sufficient tumor cell uptake and, hence, nuclear localization. Here, describe a de novo computational approach for generating 43 novel NLSs (based on 3 different NLS classes) that contained amino acid substitutions to achieve net-neutral charge states. We also introduce a novel nuclear isolation-quantitative flow cytometric method for determining nuclear localization efficiency. Material and methods An algorithm was created based on complete interaction binding affinity strengths for 20 x 20 pairs of octapeptides consisting of the 20 common amino acids. 9 PDB files were selected as templates and were comprised of viral, RNA processing, and transcription initiation protein NLSs bound to Impa. In silico alanine scanning on all NLS templates generated rankings on amino acid sensitivities for each position in the sequences. Non-sensitive residues were then subjected to residue scanning for generating net-neutral charged NLSs. Computational docking studies generated predictive binding scores relative to wild type NLSs. Favorable NLS candidates were genetically fused to GFP. In contrast to utilizing fluorescence microscopy, which cannot determine nuclear localization efficiency, we created a method to isolate nuclei from transiently transfected CHOK1 cells and quantify nuclear localization by flow cytometry. Results 2-8 net-neutral NLSs with good binding for Imga could be generated for each PDB file. The net-neutral mutants often contained an abundance of glutamic and/or aspartic acid substitutions, and were able to bind to Impa residues to compensate for the replaced amino acids. Transfected GFP-NLS constructs displayed variable fluorescence expression kinetics. Therefore, we created an in-house plasma membrane lysis protocol to isolate intact nuclei. Quantitative evaluations are currently underway by evaluating nuclei fluorescence by flow cytometry at various time points. Thus far, the tested GFP-NLSs are able to localize to the nucleus. Conclusions An important objective of computational protein design is the generation of high affinity peptides as a precursor to the development of therapeutics, and as a tool to aid researchers in understanding governing interaction principles of specific complexes. We have achieved both the development of potential NLS peptides for overcoming the cationic-sequestration barrier, and for understanding novel NLS principles to further advance the NLS-therapeutics field. Citation Format: Olga Bednova, Alexis Rioux-Chevalier, Dipika Patel, Mathieu Boudreau, Jeffrey Victor Leyton. De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P061.