N-terminal Variants Research Articles

Heterozygous variants in the teashirt zinc finger homeobox 3 (TSHZ3) gene in human congenital anomalies of the kidney and urinary tract.

Around 180 genes have been associated with congenital anomalies of the kidney and urinary tract (CAKUT) in mice, and represent promising novel candidate genes for human CAKUT. In whole-exome sequencing data of two siblings with genetically unresolved multicystic dysplastic kidneys (MCDK), prioritizing variants in murine CAKUT-associated genes yielded a rare variant in the teashirt zinc finger homeobox 3 (TSHZ3) gene. Therefore, the role of TSHZ3 in human CAKUT was assessed. Twelve CAKUT patients from 9/301 (3%) families carried five different rare heterozygous TSHZ3 missense variants predicted to be deleterious. CAKUT patients with versus without TSHZ3 variants were more likely to present with hydronephrosis, hydroureter, ureteropelvic junction obstruction, MCDK, and with genital anomalies, developmental delay, overlapping with the previously described phenotypes in Tshz3-mutant mice and patients with heterozygous 19q12-q13.11 deletions encompassing the TSHZ3 locus. Comparable with Tshz3-mutant mice, the smooth muscle layer was disorganized in the renal pelvis and thinner in the proximal ureter of the nephrectomy specimen of a TSHZ3 variant carrier compared to controls. TSHZ3 was expressed in the human fetal kidney, and strongly at embryonic day 11.5-14.5 in mesenchymal compartments of the murine ureter, kidney, and bladder. TSHZ3 variants in a 5' region were more frequent in CAKUT patients than in gnomAD samples (p < 0.001). Mutant TSHZ3 harboring N-terminal variants showed significantly altered SOX9 and/or myocardin binding, possibly adversely affecting smooth muscle differentiation. Our results provide evidence that heterozygous TSHZ3 variants are associated with human CAKUT, particularly MCDK, hydronephrosis, and hydroureter, and, inconsistently, with specific extrarenal features, including genital anomalies.

Altered O-glycosylation of β1-adrenergic receptor N-terminal single-nucleotide variants modulates receptor processing and functional activity.

N-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human β1-adrenergic receptor (β1AR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O-glycosylation assays, analysis of native receptor N-terminal O-glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O-glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O-glycosites that enhanced or inhibited cleavage at the adjacent sites (P52↓L53 and R31↓L32), respectively, or abolished the major site at R31↓L32 (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full-length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol-mediated signaling in an enhanced bystander bioluminescence energy transfer β-arrestin2 recruitment assay in a coordinated manner with the common C-terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a β1AR gain-of-function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

Detection and Evaluation of Procalcitonin Variants As Diagnostic Tools in Systemic Inflammation

BACKGROUND: Procalcitonin is an indicator of systemic inflammation associated with major surgery or sepsis. Procalcitonin exists in a full-length and truncated variant as a result of dipeptidylpeptidase-4 (DPP4)-cleavage. We recently identified differential biological activity of both variants. Here, we present an immunoassay-based method for the separate detection of procalcitonin variants and correlation to clinical data in patients with severe systemic inflammation. METHODS: Rabbits were immunized with peptides of N-terminal sequences of both human procalcitonin variants and polyclonal antibodies purified from rabbit plasma. Antibodies were used for the detection of procalcitonin variants in an indirect sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available monoclonal anti-procalcitonin antibody as capture. Serum was collected from 19 septic patients exhibiting hyperprocalcitonemia as part of a cross-sectional study; clinical data were analyzed and correlated with procalcitonin variant measurements. DPP4 activity was determined by a DPP4 activity assay. RESULTS: Purified antibodies allowed for the separate detection of both procalcitonin variants in all patients. Levels of truncated procalcitonin (truncPCT) correlated with DPP4-activity (Pearson’s R = 0.85, P &lt; .001) and negatively correlated with patients’ Sequential Organ Failure Score (SOFA) scores (Pearson’s R = –0.56, P = .013). In contrast, the correlation between full-length procalcitonin (fullPCT) and SOFA scores was positive (Pearson’s R = 0.56, P = .013). Separation of the patient collective into groups with higher amounts of fullPCT versus truncPCT revealed higher SOFA scores in patients with fullPCT &gt; truncPCT (mean ± standard error of the mean; 11. 3 ± 0.8 vs 6. 1 ± 1.5, P = .003). Patients with fullPCT &gt; truncPCT showed a tendency towards higher doses of vasopressor (0. 2 ± 0.1 vs 0. 1 ± 0.03 µg/kg/min norepinephrine within the first 24 hours after sepsis diagnosis, P = .062) and exhibited higher creatinine (2. 0 ± 0.2 vs 1. 4 ± 0.3mg/dL, P = .019) and leukocyte levels (31. 0 ± 5.4 vs 12. 8 ± 1.9cells/µL, P = .012). In addition, patients with fullPCT &gt; truncPCT were more often subjected to treatment with hydrocortisone (49.0 vs 0%, P = .018). CONCLUSIONS: Polyclonal antibodies generated using procalcitonin N-terminal variant peptides as immunogens are suitable for procalcitonin variant assessment. The separate detection of procalcitonin variants may offer additional diagnostic value and can be correlated with organ dysfunction and clinical outcomes in patients with systemic inflammation.

Comprehensive EHMT1 variants analysis broadens genotype-phenotype associations and molecular mechanisms in Kleefstra syndrome

The shift to a genotype-first approach in genetic diagnostics has revolutionized our understanding of neurodevelopmental disorders, expanding both their molecular and phenotypic spectra. Kleefstra syndrome (KLEFS1) is caused by EHMT1 haploinsufficiency and exhibits broad clinical manifestations. EHMT1 encodes euchromatic histone methyltransferase-1-a pivotal component of the epigenetic machinery. We have recruited 209 individuals with a rare EHMT1 variant and performed comprehensive molecular in silico and invitro testing alongside DNA methylation (DNAm) signature analysis for the identified variants. We (re)classified the variants as likely pathogenic/pathogenic (molecularly confirming Kleefstra syndrome) in 191 individuals. We provide an updated and broader clinical and molecular spectrum of Kleefstra syndrome, including individuals with normal intelligence and familial occurrence. Analysis of the EHMT1 variants reveals a broad range of molecular effects and their associated phenotypes, including distinct genotype-phenotype associations. Notably, we showed that disruption of the "reader" function of the ankyrin repeat domain by a protein altering variant (PAV) results in a KLEFS1-specific DNAm signature and milder phenotype, while disruption of only "writer" methyltransferase activity of the SET domain does not result in KLEFS1 DNAm signature or typical KLEFS1 phenotype. Similarly, N-terminal truncating variants result in a mild phenotype without the DNAm signature. We demonstrate how comprehensive variant analysis can provide insights into pathogenesis of the disorder and DNAm signature. In summary, this study presents a comprehensive overview of KLEFS1 and EHMT1, revealing its broader spectrum and deepening our understanding of its molecular mechanisms, thereby informing accurate variant interpretation, counseling, and clinical management.

P-268 Unique Argonaute isoforms from mammalian oocytes: unveiling their true purpose

Abstract Study question How does the mouse oocyte-specific AGO2 isoform (AGO2ooc) affect RNA interference (RNAi) and microRNA (miRNA) pathways? Summary answer Mouse AGO2ooc presents reduced RNAi-like endonucleolytic cleavage of perfectly complementary targets. Functional analysis of its impact on fertility and oocyte transcriptome control is in progress. What is known already Argonaute proteins play a pivotal role in effector complexes dedicated to miRNA and RNAi pathways, where small RNAs mediate sequence-specific repression of target RNAs. While all mammalian AGO proteins (AGO1 to AGO4) can function in the miRNA pathway, inhibiting target translation, AGO2 uniquely engages in the RNAi pathway, directing the endonucleolytic cleavage of targets perfectly complementary to small RNAs. In mammalian somatic cells, miRNAs are part of a crucial posttranscriptional regulation network, while RNAi is mostly non-functional. Mouse oocytes, however, deviate from this norm: miRNA regulation proves dispensable, while the abrogation of RNAi results in meiotic spindle defects and sterility. Study design, size, duration Experimental study. Participants/materials, setting, methods We pooled 25 fully-grown GV oocytes in triplicates from non-stimulated 10-12 weeks-old C57BI/6NCrl females for RNA isolation and sequencing. To assess RNAi-like and miRNA-like activity, we transfected Ago2ooc/ooc clones and WT cells with NanoLuc luciferase reporters bearing one perfect complementary (1xP, RNAi-like), four bulged (4xB, miRNA-like) or four mutated (4xM, control) sites for Let-7a and miR-30c. A firefly luciferase reporter without any site was co-transfected for normalization purposes. Main results and the role of chance RNA sequencing revealed an alternative exon-1 in mouse oocyte AGO2, producing an isoform (AGO2ooc) with different amino acid composition in the N-terminus. Interestingly, the somatic version of this N-terminus presents amino acid residues known to be post-translationally modified. These residues are absent in AGO2ooc. To functionally analyze the oocyte-specific N-terminus, we genetically engineered NIH3T3 fibroblasts to express the oocyte alternative exon-1 (Ago2ooc/ooc). Notably, AGO2ooc-expressing cells presented reduced RNAi-like (2-5 fold) but indistinguishable miRNA-like activity when compared to WT cells in reporter assays for Let-7a and miR-30c. The lower RNAi-like activity presented by AGO2ooc-expressing cells was further confirmed in an independent RNAi assay employing the expression of dsRNA hairpins. Both isoforms showed similar abundance in fibroblasts and similar cleavage activity in vitro. Next, we wondered whether AGO2ooc also displays reduced RNAi activity in vivo. To answer that, we generated mouse models whose oocytes would express the somatic AGO2 isoform instead of the AGO2ooc, or N-terminal variants with mutations in putative key amino acid residues. These models will be further utilized to assess fertility and oocyte transcriptome. Limitations, reasons for caution We must investigate whether AGO2ooc also displays differential RNAi activity in mouse oocytes and is relevant for oocyte competence, using the developed mouse models. Wider implications of the findings Mouse oocytes are known to exceptionally operate RNAi. Remarkably, AGO2, the effector nuclease associated with RNAi-like target cleavage has a different N-terminus in oocytes, suggesting that this factor could have adapted to this atypical regulation. We hypothesize that AGO2ooc controls RNAi repression, gene expression and oocyte competence. Trial registration number not applicable

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

The Use of Mass Spectrometry in Therapeutic Protein Biologics License Applications: A Retrospective Review Revisited.

Biologic license applications (BLAs) for 93 therapeutic proteins approved between 2016 and 2020 were analyzed for use of mass spectrometry (MS) as a follow up to a previous study that assessed MS use in BLAs from 2000 to 2015. Thirty percent of these BLAs were biosimilars, while only one biosimilar BLA was approved prior to 2016. This analysis evaluated the use of a variety of MS techniques and instrumentation. Results were further interpreted based on the relationship of MS use over time, between drug types, and between new drugs and biosimilars. MS data were included in 93 BLAs examined. The top eight quality attributes most assessed by MS in rank order were amino acid sequence, molecular mass, oxidation, disulfide bonds, deamidation, glycosylation, N-terminal sequence variants, and C-terminal sequence variants. These attributes were the same top attributes seen previously from BLAs approved between 2000 and 2015, and the use of MS to analyze them generally continued to increase across the new time frame. The average number of attributes analyzed by MS per BLA also continued to increase over the extended time frame of 21 years. High-resolution, accurate mass instrumentation such as the Orbitrap and time-of-flight (TOF) usage increased over time for all assessed attributes, while matrix-assisted laser desorption/ionization (MALDI)-TOF/(TOF) usage decreased. From highest to lowest rank, the top 11 attributes were antibody drug conjugate (ADC) characterization (i.e., drug load distribution/drug to antibody ratio (DAR), ADC and linkage site, and synthetic linker), isomerization, folding/higher-order structure (HOS), truncation, host cell proteins (HCPs), sequence variants (amino acid substitutions), succinimidation, glycation, PEGylation, charge variants, and oxidation.

Lack of evidence for coevolution between oxytocin receptor N-terminal variants and monogamy in placental mammals

Oxytocin (OXT) is a neurohypophyseal hormone that influences a wide range of affiliative behaviors, such as pair-bonding and infant care, across mammals. The effects of OXT depend significantly on an adequate interaction with its receptor, OXTR. OXTR belongs to the G-protein coupled receptor family. The extracellular N-terminal domain of OXTR interacts with the linear C-terminal tail of OXT and is required for OXT binding. Across mammalian species there is a genetic diversity in OXTR terminal sequence. Previous work on primates has shown an association between OXTR phylogeny and monogamy. However, it is not clear whether this variation coevolved with either mating system (monogamy) or infant care behaviors (such as allomaternal care). Here, we take a phylogenetic comparative and evolutionary modeling approach across a wide range of placental mammals (n = 60) to test whether OXTR N-terminal variants co-evolved with either monogamy or allomaternal care behaviors. Our results indicate that the diversity in OXTR N-terminal region is unlikely to provide the underlying genetic bases for variation in mating system and/or allomaternal behavior as we find no evidence for co-evolution between protein sequence and affiliative behaviors. Hence, the role played by OXT in influencing affiliative behaviors is unlikely to be mediated by the genetic diversity of its receptor.

Characterization of N-Terminal Asparagine Deamidation and Clipping of a Monoclonal Antibody.

This study presents a novel degradation pathway of a human immunoglobulin G (IgG) molecule featuring a light chain N-terminal asparagine. We thoroughly characterize this pathway and investigate its charge profiles using cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Beyond the well-documented asparagine deamidation into isoaspartic acid, aspartic acid, and succinimide intermediate, a previously unreported clipping degradation pathway is uncovered. This newly identified clipped N-terminal IgG variant exhibits a delayed elution in CEX, categorized as a "basic variant", while retaining the same main peak isoelectric point (pI) in cIEF. The influence of temperature and pH on N-terminal asparagine stability is assessed across various stressed conditions. A notable correlation between deamidation percentage and clipped products is established, suggesting a potential hydrolytic chemical reaction underlying the clipping process. Furthermore, the impact of N-terminal asparagine modifications on potency is evaluated through ELISA binding assays, revealing minimal effects on binding affinity. Sequence alignment reveals homology to a human IgG with the germline gene from Immunoglobulin Lambda Variable 6-57 (IGLV6-57), which has implications for amyloid light-chain (AL) amyloidosis. This discovery of the N-terminal clipping degradation pathway contributes to our understanding of immunoglobulin light chain misfolding and amyloid fibril deposition under physiological conditions.

Identification of a novel germline APC N-terminal pathogenic variant associated with attenuated familial adenomatous polyposis

Identification of a novel germline APC N-terminal pathogenic variant associated with attenuated familial adenomatous polyposis

Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals

PurposeCoffin-Siris and Nicolaides-Baraitser syndromes are recognizable neurodevelopmental disorders caused by germline variants in BAF complex subunits. The SMARCC2 BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort. MethodsClinical symptoms for 41 novel and 24 previously published affected individuals were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlations, molecular data were standardized and grouped into non-truncating and likely gene-disrupting (LGD) variants. Missense variant protein expression and BAF-subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays. ResultsNeurodevelopmental delay with intellectual disability, muscular hypotonia, and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, whereas non-truncating variants were mostly de novo and presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms. In vitro testing showed decreased protein expression for N-terminal missense variants similar to LGD. ConclusionThis study improved SMARCC2 variant classification and identified discernible SMARCC2-associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches.

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

New syndromic combined immunodeficiency with severe neurodevelopmental defects caused by biallelic null variants in the PPM1D gene

PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D) is a phosphatase that targets proteins related to the DNA damage repair system. C-terminal variants in PPM1D have been associated with: i) malignancy as somatic variants; and ii) Jansen-de-Vries (JdV) syndrome as germline heterozygous variants. JdV syndrome curses with moderate neurodevelopmental alterations and behavioral problems, such as autistic spectrum disorders (ASD), without reports of immunodeficiency.We describe two siblings (9 and 11 years old) born to consanguineous parents with progressive B and NK cell lymphopenia, agammaglobulinemia, non-ulcerative colitis and severe neurodevelopmental defects including microcephaly, severe encephalopathy, profound cognitive deficiency, and refractory epilepsy. Exome analysis revealed a homozygous N-terminal truncating variant in PPM1D (p.Thr174fs*6) in the two siblings inherited from their healthy heterozygous parents. The siblings’ clinical phenotype was markedly more severe than in the reported JdV patients, suggesting a different mechanism of action. To test this, we included a pediatric patient with classical JdV syndrome carrying an heterozygous PPM1D variant (p. Ser403fs*30). Using primary cells and exogenous overexpression we showed that mutation p.T174fs*6 abrogated protein expression by increased proteasome degradation, leading to no protein expression in a homozygous patient and a reduction in a haplosufficient parent. In contrast, the JdV variant (p.Ser403fs*30) led to accumulation of the truncated protein with conserved phosphatase activity, resulting in a previously undescribed gain-of-function (GOF) mechanism.We also demonstrated that chemical inhibition of WT-PPM1D in-vitro dramatically decreased B cell viability. This data, similar to the reported B cell deficiency in PPM1D-KO mice, helps to address the humoral deficiency in the patients, while not observed in the heterozygous parents nor JdV patient. To further explore NK cell deficiency, we will evaluate PPM1D-KO mice. We have generated EBV-B cell-derived iPSCs from PPM1D-null, PPM1D-haplosufficient an GOF-JdV individuals, to generate artificial cerebral organoids that will allow us to explore PPM1D gene-dosage effect in neurodevelopment and its potential manipulation.Here we report a novel biallelic PPM1D-null deficiency impairing B cell development. Besides, PPM1D dysregulation either by no protein expression or GOF appears to cause neurodevelopment abnormalities. Future studies will help to understand pathophysiology of PPM1D and better targeted clinical management.PPM1D research interest group:JF Quesada1, Mercè Bolasell2, Julie E. Niemela3, Jennifer L. Stoddard3, Agustin Gil Silva3, Brigette Boast3, José R Regueiro4, Pierre Martine5, David Lopez Perez5, Ettore Appella5, Laia Alsina6,7,8, Harry L Malech9, Hye Sun Kuehn3, Sergio D. Rosenzweig31. Department of Genetics, Hospital Universitario 12 de Octubre, Madrid, Spain.2. Department of Genetic and Molecular Medicine IPER, Institut de Recerca, Hospital Sant Joan de Déu Barcelona, Barcelona, Spain.3. Immunology Service, Department of Laboratory Medicine, Clinical Center, NIH, Bethesda, Maryland, USA4. Department of Immunology, Complutense University School of Medicine and Hospital 12 de Octubre Health Research Insitute, Madrid, Spain5. Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.6. Clinical Immunology and Primary Immunodeficiencies Unit. Allergy and Clinical Immunology Department. Hospital Sant Joan de Déu. Barcelona, Spain.7. Clinical Immunology Unit. Hospital Sant Joan de Déu-Hospital Clinic. Barcelona, Spain.8. Study Group for Immune Dysfunction Diseases in Children (GEMDIP). Institut de Recerca Sant Joan de Déu. Barcelona, Spain.9. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD.

Abstract 3735: N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation

Abstract Cancer is one of the most dreaded diseases of the 21st century for humans. Currently, the human cancer mortality rate is approximately 11-25%. However, for some animals, such as elephants, the cancer mortality rate is lower than 5%. p53 is a significant cellular regulator in human cancers, and its important role in response to DNA damage has been highlighted by the discovery of p53 retrogene found in elephants (19 copies) but not humans (0 copies). Therefore, the main question is whether (1) more copies of p53 are better at combating cancer development or (2) is elephant p53 is better than human p53 in p53-mediated function? Herein, we demonstrate for the first time that N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation. First, we created elephant-to-human p53 variants (such as T387R, L383P, LSTEL, etc.) and truncations at the N-terminus and C-terminus of human p53. Secondly, R175H and R273H p53 mutants (conformational and contact mutants, respectively) were used as negative control. Using a p53 (14X) response element LUC and several natural p53 downstream gene promoters, we demonstrated that several N-terminal and C-terminal p53 variants and truncations increases p53 transactivation compared to WT p53. Moreover, we found that deletion of the region between aa374 and aa382 is integral for p53 transactivation. Taken together, our preliminary results demonstrate that several N-terminal and C-terminal elephant-to-human p53 variants enhance p53 transactivation and possible stability. This data is the first step to support the notion that elephant p53 is better than human p53 in p53-mediated function. Citation Format: William H. Yang, Wei-Hsiung Yang. N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3735.

Backbone NMR resonance assignment of the apo human Tsg101-UEV domain.

The Endosomal Sorting Complex Required for Transport (ESCRT) pathway, through inverse topology membrane remodeling, is involved in many biological functions, such as ubiquitinated membrane receptor trafficking and degradation, multivesicular bodies (MVB) formation and cytokinesis. Dysfunctions in ESCRT pathway have been associated to several human pathologies, such as cancers and neurodegenerative diseases. The ESCRT machinery is also hijacked by many enveloped viruses to bud away from the plasma membrane of infected cells. Human tumor susceptibility gene 101 (Tsg101) protein is an important ESCRT-I complex component. The structure of the N-terminal ubiquitin E2 variant (UEV) domain of Tsg101 (Tsg101-UEV) comprises an ubiquitin binding pocket next to a late domain [P(S/T)AP] binding groove. These two binding sites have been shown to be involved both in the physiological roles of ESCRT-I and in the release of the viral particles, and thus are attractive targets for antivirals. The structure of the Tsg101-UEV domain has been characterized, using X-ray crystallography or NMR spectroscopy, either in its apo-state or bound to ubiquitin or late domains. In this study, we report the backbone NMR resonance assignments, including the proline signals, of the apo human Tsg101-UEV domain, that so far was not publicly available. These data, that are in good agreement with the crystallographic structure of Tsg101-UEV domain, can therefore be used for further NMR studies, including protein-protein interaction studies and drug discovery.

Cardioprotective role of inner membrane chloride channel, CLIC5 from ischemia-reperfusion injury.

Mitochondrial chloride channels by regulating mitochondrial volume, pH and membrane potential are important in determining the cell fate. However, very few of them are identified in mitochondria. Previously, we demonstrated the localization of a member of chloride intracellular channel (CLIC) family, CLIC5 in the inner mitochondrial membrane and its importance in regulating reactive oxygen species (ROS) generation. Herein, we show the novel role of an alternative N-terminal splice variant clic5b in cardiac mitochondrial physiology. Mass spectrometry and qPCR analysis indicated the presence of the splice variant, clic5b in cardiac tissue in addition to the prominent transcript variant, clic5a. Interestingly, the presence of splice variant clic5b was correlated with its mitochondrial localization in myoblast cells, H9C2 (55.12 ± 7.1%) but not in mouse lung epithelial cells (11 ± 2.1%). Further, using transfection studies we showed that the N terminal region of clic5b is integral for its mitochondrial targeting in neonatal cardiomyocytes and in HEK cells as transfection of clic5a does not exhibit mitochondrial localization. We also demonstrate increased mitochondrial ROS generation in clic5-/- mouse heart (N=4, p≤0.05) in comparison to wild type (wt) mice. On the other hand, lungs, that only contain splice variant clic5a, neither demonstrate mitochondrial localization of CLIC5 nor any change in ROS production. The absence of CLIC5 exacerbated myocardial infarction (52 ± 9.5% versus 38.6 ± 4.5%, n≥3, p≤ 0.05) and reduced left ventricular function upon ischemia-reperfusion (IR) injury in clic5-/- mice in comparison to wt mice suggesting a cardioprotective role of CLIC5. Furthermore, in human heart failure samples, we observed a differential expression of the splice variants clic5a and clic5b in comparison to healthy hearts. Thus, our study highlights the role of splice variant clic5b in regulating cardiac mitochondrial physiology and cardioprotection from IR injury.

Molecular determinants of the interaction between HSV-1 glycoprotein D and heparan sulfate.

Literature has well-established the importance of 3-O-sulfation of neuronal cell surface glycan heparan sulfate (HS) to its interaction with herpes simplex virus type 1 glycoprotein D (gD). Previous investigations of gD to its viral receptors HVEM and nectin-1 also highlighted the conformational dynamics of gD's N- and C-termini, necessary for viral membrane fusion. However, little is known on the structural interactions of gD with HS. Here, we present our findings on this interface from both the glycan and the protein perspective. We used C-terminal and N-terminal gD variants to probe the role of their respective regions in gD/HS binding. The N-terminal truncation mutants (with Δ1-22) demonstrate equivalent or stronger binding to heparin than their intact glycoproteins, indicating that the first 22 amino acids are disposable for heparin binding. Characterization of the conformational differences between C-terminal truncated mutants by sedimentation velocity analytical ultracentrifugation distinguished between the "open" and "closed" conformations of the glycoprotein D, highlighting the region's modulation of receptor binding. From the glycan perspective, we investigated gD interacting with heparin, heparan sulfate, and other de-sulfated and chemically defined oligosaccharides using surface plasmon resonance and glycan microarray. The results show a strong preference of gD for 6-O-sulfate, with 2-O-sulfation becoming more important in the presence of 6-O-S. Additionally, 3-O-sulfation shifted the chain length preference of gD from longer chain to mid-chain length, reaffirming the sulfation site's importance to the gD/HS interface. Our results shed new light on the molecular details of one of seven known protein-glycan interactions with 3-O-sulfated heparan sulfate.

Amyloid beta and its naturally occurring N-terminal variants are potent activators of human and mouse formyl peptide receptor 1

Formyl peptide receptors (FPRs) may contribute to inflammation in Alzheimer's disease through interactions with neuropathological Amyloid beta (Aβ) peptides. Previous studies reported activation of FPR2 by Aβ1-42, but further investigation of other FPRs and Aβ variants is needed. This study provides a comprehensive overview of the interactions of mouse and human FPRs with different physiologically relevant Aβ-peptides using transiently transfected cells in combination with calcium imaging. We observed that, in addition to hFPR2, all other hFPRs also responded to Aβ1-42, Aβ1-40, and the naturally occurring variants Aβ11-40 and Aβ17-40. Notably, Aβ11-40 and Aβ17-40 are very potent activators of mouse and human FPR1, acting at nanomolar concentrations. Buffer composition and aggregation state are extremely crucial factors that critically affect the interaction of Aβ with different FPR subtypes. To investigate the physiological relevance of these findings, we examined the effects of Aβ11-40 and Aβ17-40 on the human glial cell line U87. Both peptides induced a strong calcium flux at concentrations that are very similar to those obtained in experiments for hFPR1 in HEK cells. Further immunocytochemistry, qPCR, and pharmacological experiments verified that these responses were primarily mediated through hFPR1. Chemotaxis experiments revealed that Aβ11-40 but not Aβ17-40 evoked cell migration, which argues for a functional selectivity of different Aβ peptides. Together, these findings provide the first evidence that not only hFPR2 but also hFPR1 and hFPR3 may contribute to neuroinflammation in Alzheimer's disease through an interaction with different Aβ variants.

Two novel bombesin-like neuropeptides from the skin secretion of Pelophylax kl. esculentus: Ex vivo pharmacological characterization on rat smooth muscle types.

Mammalian bombesin-like neuropeptides (BLPs) play an important role in regulation of physiological and pathophysiological processes. Frog skin-derived BLPs, of smaller size and diverse lengths and sequences at their N-terminus, have attracted the attention of many researchers. However, these N-terminal variants and the receptors modulating their pharmacological actions are poorly studied and less understood. In this study, two BLPs, namely, [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin with primary structures NLGKQWATGHFM and NLGKQWAVGHFM were isolated from the skin secretion of hybrid Pelophylax kl. esculentus. Both BLPs share a similar primary structure with only a single amino acid substitution at the eighth position (threonine to valine), while they have quite different myotropic potencies with EC50 values in the range of 22.64 ± 9.7 nM (N = 8) to 83.93 ± 46.9 nM (N = 8). The potency of [Asn3, Lys6, Thr10, Phe13]3–14-bombesin was approximately 3-fold higher than that of [Asn3, Lys6, Phe13]3–14-bombesin. Through the investigation of receptor selectivity using a canonical bombesin receptor antagonist, it was found that [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin had an affinity to both BB1 and BB2 receptors. Their contractile functions are mainly modulated by both BB1 and BB2 receptors on rat urinary bladder and BB2 alone on rat uterus smooth muscle preparations. These data may provide new insights into the design of potent and selective ligands for bombesin receptors. Moreover, [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin did not induce significant hemolysis and toxicity in normal human cells, suggesting that these two natural novel BLPs have great potential for development into new drug candidates.

Structure of Lacticaseicin 30 and Its Engineered Variants Revealed an Interplay between the N-Terminal and C-Terminal Regions in the Activity against Gram-Negative Bacteria.

Lacticaseicin 30 is one of the five bacteriocins produced by the Gram-positive Lacticaseibacillus paracasei CNCM I-5369. This 111 amino acid bacteriocin is noteworthy for being active against Gram-negative bacilli including Escherichia coli strains resistant to colistin. Prediction of the lacticaseicin 30 structure using the Alphafold2 pipeline revealed a largely helical structure including five helix segments, which was confirmed by circular dichroism. To identify the structural requirements of the lacticaseicin 30 activity directed against Gram-negative bacilli, a series of variants, either shortened or containing point mutations, was heterologously produced in Escherichia coli and assayed for their antibacterial activity against a panel of target strains including Gram-negative bacteria and the Gram-positive Listeria innocua. Lacticaseicin 30 variants comprising either the N-terminal region (amino acids 1 to 39) or the central and C-terminal regions (amino acids 40 to 111) were prepared. Furthermore, mutations were introduced by site-directed mutagenesis to obtain ten bacteriocin variants E6G, T7P, E32G, T33P, T52P, D57G, A74P, Y78S, Y93S and A97P. Compared to lacticaseicin 30, the anti-Gram-negative activity of the N-terminal peptide and variants E32G, T33P and D57G remained almost unchanged, while that of the C-terminal peptide and variants E6G, T7P, T52P, A74P, Y78S, Y93S and A97P was significantly altered. Finally, the N-terminal region was further shortened to keep only the first 20 amino acid part that was predicted to include the first helix. The anti-Gram-negative activity of this truncated peptide was completely abolished. Overall, this study shows that activity of lacticaseicin 30, one of the rare Gram-positive bacteriocins inhibiting Gram-negative bacteria, requires at least two helices in the N-terminal region and that the C-terminal region carries amino acids playing a role in modulation of the activity. Taken together, these data will help to design forthcoming variants of lacticaseicin 30 as promising therapeutic agents to treat infections caused by Gram-negative bacilli.