Wild-type cDNA Research Articles

Primary structure of human neutrophil antigens 1a and 1b.

Neutrophil specific Fcγ receptor IIIb (CD16b) is a low-affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA-1a and HNA-1b differ in four amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA-1a, -1b epitopes is currently unknown. Permutation of each polymorphic amino acid from wild-type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well-characterized HNA antisera in an antigen capture assay. Analyzing the reaction pattern revealed that anti-HNA-1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti-HNA-1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti-HNA-1a and anti-HNA-1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind. Whereas the primary structure of HNA-1a and HNA-1b usually differs in four amino acids, epitope composition is not "antithetical". N65 alone determines the presence of HNA-1a, and S36 and/or N82 determine the presence of HNA-1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA-1 phenotype is predicted from a genotype.

Lipoid congenital adrenal hyperplasia due to steroid acute regulatory protein (STAR) variants in Three Chinese patients

Congenital adrenal hyperplasia (CAH) encompasses a group of autosomal recessive diseases characterized by enzymatic defects in steroid synthesis. Lipoid congenital adrenal hyperplasia (LCAH) is the most severe form of CAH, insofar as the initial step of steroid synthesis is impaired. Variants in the steroid acute regulatory (STAR) gene are responsible for LCAH. To describe the clinical and genetic characteristics of three Chinese patients with LCAH. We analyzed the history, clinical manifestations, physical examination, laboratory data, and computed tomography findings of three girls with LCAH. The STAR gene of the probands and their parents were sequenced using genomic DNA. The wild-type and mutant STAR cDNAs were inserted into the pcDNA3.1(+) plasmid vector and transiently transfected into COS7 cells. The enzymatic activities of the wild-type and mutant STAR were evaluated by the enzyme-dependent conversion efficiency of cholesterol to pregnenolone. We identified the molecular genetic abnormalities in three patients with LCAH. All three patients had a female phenotype: karyotype of patients 1 and 2 was 46, XY and patient 3 was 46, XX. DNA sequencing revealed compound heterozygous variants in STAR for three probands. Two variants, c.659A > G/p.His220Arg and exon 2–3 deletion, were novel. In vitro functional studies uncovered that the His220Arg variant retained 19.2 % of enzymatic activity compared to that of the wild type.

Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle

Cytochrome P450 3A is the most important CYP subfamily in humans, and CYP3A4/CYP3A5 genetic variants contribute to inter-individual variability in drug metabolism. However, no information is available for bovine CYP3A (bCYP3A). Here we described bCYP3A missense single nucleotide variants (SNVs) and evaluated their functional effects. CYP3A28, CYP3A38 and CYP3A48 missense SNVs were identified in 300 bulls of Piedmontese breed through targeted sequencing. Wild-type and mutant bCYP3A cDNAs were cloned and expressed in V79 cells. CYP3A-dependent oxidative metabolism of testosterone (TST) and nifedipine (NIF) was assessed by LC-MS/MS. Finally, SNVs functional impact on TST hydroxylation was measured ex vivo in liver microsomes from individually genotyped animals. Thirteen missense SNVs were identified and validated. Five variants showed differences in CYP3A catalytic activity: three CYP3A28 SNVs reduced TST 6β-hydroxylation; one CYP3A38 variant increased TST 16β-hydroxylation, while a CYP3A48 SNV showed enhanced NIF oxidation. Individuals homozygous for rs384467435 SNV showed a reduced TST 6β-hydroxylation. Molecular modelling showed that most of SNVs were distal to CYP3A active site, suggesting indirect effects on the catalytic activity. Collectively, these findings demonstrate the importance of pharmacogenetics studies in veterinary species and suggest bCYP3A genotype variation might affect the fate of xenobiotics in food-producing species such as cattle.

Novel compound heterozygous pathogenic variants in nucleotide-binding protein like protein (NUBPL) cause leukoencephalopathy with multi-systemic involvement

NUBPL (Nucleotide-binding protein like) protein encodes a member of the Mrp/NBP35 ATP-binding proteins family, deemed to be involved in mammalian complex I (CI) assembly process. Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL (p.Phe242Leu/p.Leu104Pro). We investigated patient's and control immortalised fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In addition, complex III stability was moderately affected as well. Lentiviral-mediated expression of the wild-type NUBPL cDNA rescued both CI and CIII assembly defects, confirming the pathogenicity of the variants. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, we confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and we demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

A Novel Human Congenital Cataract Mutation in EPHA2 Kinase Domain (p.G668D) Alters Receptor Stability and Function.

To identify the genetic defect in a four-generation Chinese family that causes autosomal dominant congenital posterior subcapsular cataracts, and to understand how this EPHA2 kinase domain mutation affects EPHA2 activity. Variants in 54 cataract-associated genes were screened by targeted next generation sequencing (NGS) and then validated by Sanger sequencing. EPHA2 wild-type cDNA was synthesized in vitro, and EPHA2 p.G668D mutant was constructed by PCR site-directed mutagenesis. Western blotting and fluorescence microscopy were used to analyze the expression level of protein and its subcellular localization, respectively. A wound-healing assay was performed to analyze changes to cell migration. A novel heterozygous missense mutation was identified in the kinase domain of the EPHA2 gene (c.2003G>A, p.G668D). This is the third congenital cataract mutation being reported in this domain. Functional study revealed that the kinase domain mutation (p.G668D) decreased EphA2 protein level (P = 0.036) via a proteasome-dependent pathway, altered its subcellular localization of the EphA2 from cell-cell contacts to a diffuse perimembranous distribution, and changed the distribution of β-catenin as well. The expression of mutant EphA2 significantly promoted the migration of human lens epithelial cells (P = 0.002). Our study presented the evidence for a novel EPHA2 kinase domain mutation that causes congenital posterior subcapsular cataracts. The first functional study on an EPHA2 kinase domain mutation that causes a congenital cataract revealed that the G668D mutation destabilized the receptor, changed its subcellular localization, and altered the activation of EphA2 with its ligand ephrin. The mutant EphA2 resulted in a reduced inhibition of cell migration. As a consequence, the c.G668D mutation promoted cell migration and caused the formation of cataracts.

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

Two Novel er1 Alleles Conferring Powdery Mildew (Erysiphe pisi) Resistance Identified in a Worldwide Collection of Pea (Pisum sativum L.) Germplasms.

Powdery mildew caused by Erysiphe pisi DC. severely affects pea crops worldwide. The use of resistant cultivars containing the er1 gene is the most effective way to control this disease. The objectives of this study were to reveal er1 alleles contained in 55 E. pisi-resistant pea germplasms and to develop the functional markers of novel alleles. Sequences of 10 homologous PsMLO1 cDNA clones from each germplasm accession were used to determine their er1 alleles. The frame shift mutations and various alternative splicing patterns were observed during transcription of the er1 gene. Two novel er1 alleles, er1-8 and er1-9, were discovered in the germplasm accessions G0004839 and G0004400, respectively, and four known er1 alleles were identified in 53 other accessions. One mutation in G0004839 was characterized by a 3-bp (GTG) deletion of the wild-type PsMLO1 cDNA, resulting in a missing valine at position 447 of the PsMLO1 protein sequence. Another mutation in G0004400 was caused by a 1-bp (T) deletion of the wild-type PsMLO1 cDNA sequence, resulting in a serine to leucine change of the PsMLO1 protein sequence. The er1-8 and er1-9 alleles were verified using resistance inheritance analysis and genetic mapping with respectively derived F2 and F2:3 populations. Finally, co-dominant functional markers specific to er1-8 and er1-9 were developed and validated in populations and pea germplasms. These results improve our understanding of E. pisi resistance in pea germplasms worldwide and provide powerful tools for marker-assisted selection in pea breeding.

THE SCN5A VARIANT C683R RESULTS IN A GAIN-OF-FUNCTION OF THE CARDIAC SODIUM CURRENT (NAV1.5) WITH ADRENERGIC MODULATION

Congenital Long QT syndrome (LQTS) is an inherited arrhythmia characterized by prolonged cardiac repolarization that may result in torsades de pointes and sudden cardiac death. Gain-of-function variants of the SCN5A gene, encoding the cardiac sodium channel Nav1.5; INa, underlie nearly 10% of LQTS and cause LQT3. In LQT3 patients, bradycardia and low sympathetic tone (associated with sleep and rest) have been considered to trigger the arrhythmias. Recently, a novel SCN5A variant (C683R) was identified in two unrelated patients with personal/family history of prolonged QT and sudden cardiac arrest. Paradoxically, C683R-associated arrhythmias in these two patients have only been observed during hyperadrenergic states. We therefore conducted a functional study to characterize the electrophysiological effects of the SCN5A C683R variant on Nav1.5 current in a cellular model. The C683R SCN5A variant was engineered into the pcDNA1 vector by using site-directed mutagenesis. Wild-Type (WT) or C683R SCN5A cDNA was transfected into tsA201 cells, along with the human Nav1.5 channel ß1-subunit. Electrophysiological characterization of the C683R variant was performed using the whole cell patch-clamp technique. The selective protein kinase A (PKA) activator 8-CPT-cAMP 50 μM was used in the pipette solution to mimic the effect of epinephrine on either WT or C683R Nav1.5 currents. The C683R variant did not generate any persistent (late) Nav1.5 current when compared to WT. It did not cause any significant change on recovery from inactivation vs WT nor on both steady-state and slow inactivation. However, the C683R variant increased maximal Nav1.5 current density from 341±64 to 766±103 pA/pF when compared to WT (p < 0.05, n≥9) and the midpoint of activation voltage (V½) was negatively shifted from -57.18±1.24 to -63.63±1.40 mV (p < 0.001 vs WT). Moreover, when exposed to the PKA activator 8-CPT-cAMP 50 μM, the C683R variant showed a greater maximal Nav1.5 current density than WT; 993±100 vs 645±137 pA/pF (p < 0.05, n≥6). Our observations suggest a gain-of-function of the Nav1.5 current in the C683R variant compared to the WT protein. This gain of function may be further enhanced upon stimulation with catecholamines. The C683R variant of SCN5A results in a significant gain of function of the Nav1.5 current with increased responsiveness to adrenergic stimulation. Our observations provide a likely explanation for the unusual phenotype in carriers of the C683R variant that is characterized by epinephrine-triggered ventricular arrhythmias. The electrophysiological properties are different from conventional LQT3 and may represent a distinct inherited arrhythmia syndrome.

Negative regulation of eNOS-NO signaling by over-SUMOylation of PPARγ contributes to insulin resistance and dysfunction of vascular endothelium in rats

SUMOylation of peroxisome proliferator-activated receptor gamma (PPAR γ) plays important regulatory role in its transcriptional activity. Our recent studies in vitro found that over-SUMOylation of PPARγ, like high glucose and high fat (HG/HF), induced endothelial insulin resistance (IR). However, whether such an event occurs in rats remains unclear. Therefore, our study aimed at investigating whether PPARγ over-SUMOylation could mimic high sucrose/fat diet (HFD) to induce endothelial IR and dysfunction and explored its underlying mechanisms. Normal chow-fed rats were intravenously infected with adenoviruses carrying the wild type cDNAs encoding PPARγ, SUMO1 and PIAS1 (protein inhibitor of activated STAT1). HFD-fed rats were regarded as a positive control. Body physical and biochemical parameters, glucose tolerance and vessel function were detected. The expression and SUMOylation levels of PPARγ were measured by western blotting and co-immunoprecipitation. Our results showed that like HFD, PPARγ over-SUMOylation induced endothelial IR and dysfunction via a negative regulation of eNOS-NO pathway. More importantly, we found that PPARγ over-SUMOylation induced endogenous SUMOylation cascade and exacerbated endothelial IR and dysfunction.The findings will deepen the understanding on PPARγ SUMOylation-regulating insulin signaling network and offer a potential target for prevention and cure of diabetic vascular complications.

Tumor cell IDO1 increases intratumoral immunosuppressive Tregs independent of enzyme activity

Abstract Objective Indoleamine 2, 3 dioxygenase 1 (IDO1) is a rate limiting enzyme that converts tryptophan (Trp) into kynurenine (Kyn). IDO1 knockdown in murine glioblastoma (GBM) cells suppresses intratumoral regulatory T cell (Treg) recruitment and increases survival, but unexpectedly, has no effect on intratumoral Trp levels. Moreover, while GBM cell IDO1 overexpression increases intratumoral Treg levels, treatment with a pharmacological IDO1 enzyme inhibitor fails to reverse this effect. These novel data led us to question whether GBM cell IDO1 recruits Tregs independent of enzyme activity, in vivo. Methods Ido1−/− mouse GBM cells reconstituted with wild-type or enzyme-null (H350A) murine Ido1 cDNA, and IDO1−/− human GBM cells were created. HPLC for Trp/Kyn levels, cell proliferation, cell viability, and splenic macrophage-GBM cell co-cultures, were studied in vitro. Syngeneic or humanized mice intracranially-engrafted with modified mouse or human GBM cells, respectively, were studied for the effect(s) on Treg accumulation and/or survival. Results As compared with IDO1 WT GBM cells, IDO1 H350A GBM cells show a significant decrease of Trp metabolism without affecting cell proliferation and viability. As compared with vector control GBM cells, co-culture for both IDO1 WT and H350A GBM cells induces greater numbers of mature macrophages. Independent of enzyme activity, GBM cell IDO1 expression increases intratumoral Tregs and decreases animal survival. Conclusions We created novel GBM models for elucidating the mechanism of metabolism-independent, IDO1-mediated intratumoral Treg accumulation. Given the majority of cancers with IDO1 expression, we suspect that our work may be generalizable to many types of malignancies.

Use of PTC124 for nonsense suppression therapy targeting BMP4 nonsense variants in vitro and the bmp4st72 allele in zebrafish.

Nonsense suppression therapy (NST) utilizes compounds such as PTC124 (Ataluren) to induce translational read-through of stop variants by promoting the insertion of near cognate, aminoacyl tRNAs that yield functional proteins. We used NST with PTC124 to determine if we could successfully rescue nonsense variants in human Bone Morphogenetic Protein 4 (BMP4) in vitro and in a zebrafish bmp4 allele with a stop variant in vivo. We transfected 293T/17 cells with wildtype or mutant human BMP4 cDNA containing p.Arg198* and p.Glu213* and exposed cells to 0–20 μM PTC124. Treatment with 20 μM PTC124 produced a small, non-significant increase in BMP4 when targeting the p.Arg198* allele, but not the p.Glu213* allele, as measured with an In-cell ELISA assay. We then examined the ability of PTC124 to rescue the ventral tail fin defects associated with homozygosity for the p.Glu209* allele of bmp4 (bmp4st72/st72) in Danio rerio. We in-crossed bmp4st72/+ heterozygous fish and found a statistically significant increase in homozygous larvae without tail fin and ventroposterior defects, consistent with phenotypic rescue, after treatment of dechorionated larvae with 0.5 μM PTC124. We conclude that treatment with PTC124 can rescue bmp4 nonsense variants, but that the degree of rescue may depend on sequence specific factors and the amount of RNA transcript available for rescue. Our work also confirms that zebrafish show promise as a useful animal model for assessing the efficacy of PTC124 treatment on nonsense variants.

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Essentials Loss of fibrinogen in zebrafish has been previously shown to result in adult onset hemorrhage Hemostatic defects were discovered in early fga-/- embryos but well tolerated until adulthood Afibrinogenemia and thrombocytopenia results in synthetic lethality in zebrafish. Testing human FGA variants of uncertain significance in zebrafish identified causative mutations SUMMARY: Background Mutations in the alpha chain of fibrinogen (FGA), such as deficiencies in other fibrinogen subunits, lead to rare inherited autosomal recessive hemostatic disorders. These range from asymptomatic to catastrophic life-threatening bleeds and the molecular basis of inherited fibrinogen deficiencies is only partially understood. Zinc finger nucleases have been used to produce mutations in zebrafish fga, resulting in overt adult-onset hemorrhage and reduced survival. Objectives To determine the age of onset of hemostatic defects in afibrinogenemic zebrafish and model human fibrinogen deficiencies. Methods TALEN genome editing (transcription activator-like effector nucleases) was used to generate a zebrafish fga mutant. Hemostatic defects were assessed through survival, gross anatomical and histological observation and laser-induced endothelial injury. Human FGA variants with unknown pathologies were engineered into the orthologous positions in zebrafish fga. Results Loss of Fga decreased survival and resulted in synthetic lethality when combined with thrombocytopenia. Zebrafish fga mutants exhibit a severe hemostatic defect by 3 days of life, but without visible hemorrhage. Induced thrombus formation through venous endothelial injury was completely absent in mutant embryos and larvae. This hemostatic defect was restored by microinjection of wild-type fga cDNA plasmid or purified human fibrinogen. This system was used to determine whether unknown human variants were pathological by engineering them into fga. Conclusions These studies confirm that loss of fibrinogen in zebrafish results in the absence of hemostasis from the embryonic period through adulthood. When combined with thrombocytopenia, zebrafish exhibit synthetic lethality, demonstrating that thrombocytes are necessary for survival in response to hemorrhage.

Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium.

During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin-resistant T-lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild-type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T-cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real-time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470-493, 2019. © 2019 Wiley Periodicals, Inc.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

A homozygous SCN5A mutation associated with atrial standstill and sudden death.

Atrial standstill is an arrhythmogenic condition characterized by the absence of spontaneous electrical and mechanical atrial activity or in response to stimulation. There are few reported familial cases which have been associated with SCN5A mutations cosegregating with GJA5 or RYR2; however, isolated SCN5A mutations are rare. The purpose of this study was to determine the clinical and biophysical consequence of a novel SCN5A mutation identified in a family with progressive atrial standstill and sudden death. The family of a sporadic case of congenital atrial standstill underwent genetic screening. Human Embryonic Kidney 293 cells were transfected with wild-type (WT) or mutant SCN5A cDNAs. Biophysical properties were studied using whole-cell using patch clamp methods. A novel homozygous SCN5A mutation, p.V1340L, was identified in the proband and her sister. The proband had complete atrial standstill whereas the sister had partial atrial standstill. Heterozygous mutations were identified in the mother, father, and brother. All three had normal sinus rhythm and were asymptomatic. The mutant Nav1.5(V1340L) reduced Nav1.5 current density as well as showed a depolarizing shift in the voltage-dependent steady-state activation (WT: -35.3 ± 1.62mV; V1340L: -22.4 ± 2.59mV; P=0.001). A homozygous loss-of-function SCN5A mutation likely results in atrial standstill and sudden death due to suppression of initiation of action potential.

Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface

BackgroundMonogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like receptor family CARD-containing 4 protein (NLRC4).ObjectiveHere we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. Methods: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1β/IL-18 were quantified by using flow cytometry and ELISA, respectively.ResultsThe p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1–dependent cell death, and IL-1β/IL-18 production. ASC contributed to p.W655C NLRC4–mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex.ConclusionThis is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex.

Nanocomplex-based TP53 gene therapy promotes anti-tumor immunity through TP53- and STING-dependent mechanisms

ABSTRACTLoss or mutation of TP53 has been linked to alterations in anti-tumor immunity as well as dysregulation of cell cycle and apoptosis. We explored immunologic effects and mechanisms following restoration of wild-type human TP53 cDNA in murine oral cancer cells using the therapeutic nanocomplex scL-53. We demonstrated scL-53 induces dose-dependent expression of TP53 and induction of apoptosis and immunogenic cell death. We further demonstrated both TP53-dependent and independent induction of tumor cell immunogenicity through the use of blocking mAbs, nanocomplex loaded with DNA plasmid with or without TP53 cDNA, empty nanocomplex and siRNA knockdown techniques. TP53-independent immune modulation was observed following treatment with nanocomplex loaded with DNA plasmid lacking TP53 cDNA and abrogated in STING-deficient tumor cells, supporting the presence of a cytoplasmic DNA sensing, STING-dependent type-I IFN response. Cooperatively, TP53- and STING-dependent alterations sensitized tumor cells to CTL-mediated lysis, which was further enhanced following reversal of adaptive immune resistance with PD-1 mAb. In vivo, combination scL-53 and PD-1 mAb resulted in growth control or rejection of established tumors that was abrogated in mice depleted of CD8+ cells or in STING deficient mice. Cumulatively, this work demonstrates 1) a direct anti-tumor effects of functional TP53; 2) non-redundant TP53- and STING-dependent induction of tumor cell immunogenicity following scL-53 treatment; and 3) that adaptive immune resistance following scL-53 treatment can be reversed with PD-based immune checkpoint blockade, resulting in the rejection or control of syngeneic murine tumors. These data strongly support the clinical combination of scL-53 and immune checkpoint blockade.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

A hERG mutation E1039X produced a synergistic lesion on IKs together with KCNQ1-R174C mutation in a LQTS family with three compound mutations

Congenital long QT syndrome (LQTS) caused by compound mutations is usually associated with more severe clinical phenotypes. We identified a LQTS family harboring three compound mutations in different genes (KCNQ1-R174C, hERG-E1039X and SCN5A-E428K). KCNQ1-R174C, hERG-E1039X and SCN5A-E428K mutations and/or relevant wild-type (WT) cDNAs were respectively expressed in mammalian cells. IKs-like, IKr-like, INa-like currents and the functional interaction between KCNQ1-R174C and hERG-E1039X channels were studied using patch-clamp and immunocytochemistry techniques. (1) Expression of KCNQ1-R174C alone showed no IKs. Co-expression of KCNQ1-WT + KCNQ1-R174C caused a loss-of-function in IKs and blunted the activation of IKs in response to isoproterenol. (2) Expression of hERG-E1039X alone and co-expression of hERG-WT + hERG-E1039X negatively shifted inactivation curves and decelerated the recovery time from inactivation. (3) Expression of SCN5A-E428K increased peak INa, but had no effect on late INa. (4) IKs and IKr interact, and hERG-E1039X caused a loss-of-function in IKs. (5) Immunocytochemical studies indicated that KCNQ1-R174C is trafficking defective and hERG-E1039X is defective in biosynthesis/degradation, but the abnormities were rescued by co-expression with WT. Thus, KCNQ1-R174C and hERG-E1039X disrupted IKs and IKr functions, respectively. The synergistic lesion, caused by KCNQ1-R174C and hERG-E1039X in IKs, is very likely why patients showed more severe phenotypes in the compound mutation case.