Genetic analysis of a child with severe intellectual disability caused by a novel variant in the FERM domain of the FRMPD4 protein.

Intellectual developmental disorder, X-linked 104 (XLID104), caused by the FRMPD4 gene variant, is a rare X-linked genetic disease that primarily manifests as intellectual disability (ID) and language delay, and may be accompanied by behavioural abnormalities. Currently, only 11 patients from four families have been reported to carry FRMPD4 gene variants. Here, we report a rare case of a Chinese patient with XLID104 who was presented with severe ID and language impairment. Genetic testing results showed that the patient had a novel hemizygous variant on FRMPD4 inherited from the heterozygous variant NM_001368397: c.1772A>C (p.Glu591Ala) carried by his mother. To our knowledge, this variant has not been reported previously. Western blot results for the recombinant plasmid constructed in vitro indicated that the expression of the mutant protein may be reduced. Using molecular dynamics simulations, we predicted that the mutant protein may affect the interaction of the FRMPD4 protein with DLG4. In this study, we expand the spectrum of FRMPD4 variants and suggest that the clinical awareness of the genetic diagnosis of nonsyndromic ID should be strengthened.

Mutant huntingtin protein induces MLH1 degradation, DNA hyperexcision, and cGAS-STING-dependent apoptosis.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

Transactivation by partial function P53 family mutants is increased by the presence of G-quadruplexes at a promoter site.

The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.

Impact of PmrB mutations on clinical Klebsiella pneumoniae with variable colistin-susceptibilities: Structural insights and potent therapeutic solutions.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections continue to impose high morbidity threats to hospitalized patients worldwide, limiting therapeutic options to last-resort antibiotics like colistin. However, the dynamic genomic landscape of colistin-resistant K. pneumoniae (COLR-Kp) invoked ardent exploration of underlying molecular signatures for therapeutic propositions/designs. We unveiled the structural impact of the widespread and emerging PmrB mutations involved in colistin resistance (COLR) in K. pneumoniae. In the present study, clinical isolates of K. pneumoniae expressed variable susceptibilities to colistin (>0.5 µg/mL for resistant and ≤0.25 µg/mL for susceptible) despite mutations such as T157P, G207D and T246A. The protein sequences extracted from in-house sequenced genomes were used to model mutant PmrB proteins and analyze the underlying structural alterations. The mutations were contrasted based on molecular dynamics simulation trajectories, free-energy landscapes and structural flexibility profiles. The altered backbone flexibilities can be an essential factor for mutant selection by COLR K. pneumoniae and can provide clues to deal with emerging mutants. Furthermore, PmrB having high druggability confidence (>0.99), was explored as a potential target for 1396 virtually screened FDA-approved drug candidates. Among the top-10 compounds (scores >70), amphotericin B was found to be potential candidate with high affinity (Binding energy <-8 kcal/mol) and stable interactions (RMSF <0.7 Å) against PmrB druggable pockets, despite the mutations, which encourages future adjunct therapeutic research against COLR-Kp.

OmeDDG: Improved Protein Mutation Stability Prediction Based on Predicted 3D Structures.

Determining changes in the protein's thermal stability following mutations is critical in protein engineering and understanding pathogenic missense mutations. Despite the development of various computational methods to predict the effects of single-point mutations, their accuracy remains limited. In this study, we propose a new computational method, OmeDDG, that more accurately predicts mutation-induced Gibbs free energy changes in protein folding (ΔΔG). OmeDDG takes the sequences of wild-type and mutant proteins as input, utilizes OmegaFold to obtain the 3D structure, employs a convolutional neural network to extract structural features, and combines them with protein mutation features and pretraining features to predict the stability of single-point mutations in proteins. We performed a comprehensive comparison between OmeDDG and other available prediction methods on four blind test datasets, confirming that OmeDDG can effectively enhance protein mutation prediction performance. Notably, on the antisymmetric dataset Ssym, OmeDDG achieves the best performance, demonstrating favorable antisymmetry with PCC = 0.79 and RMSE = 0.96 for forward mutations and PCC = 0.77 and RMSE = 0.97 for reverse mutant types.

Splicing mutations in AMELX and ENAM cause amelogenesis imperfecta.

BACKGROUND: Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. METHODS: Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. RESULTS: The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. CONCLUSIONS: In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation.

The extracellular matrix dictates regional competence for tumour initiation.

The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.

Association of IL-17F rs2397084 (E126G), rs11465553 (V155I) and rs763780 (H161R) variants with rheumatoid arthritis and their effects on the stability of protein.

Interleukin-17F (IL-17F), considered a pro-inflammatory cytokine, has been shown to contribute to skeletal tissue degradation and hence chronic inflammation in rheumatoid arthritis (RA). In this study we utilized bioinformatics tools to analyze the effect of three exonic SNPs (rs2397084, rs11465553, and rs763780) on the structure and function of the IL-17F gene, and evaluated their association with RA in Pakistani patients. The predicted deleterious and damaging effects of identified genetic variants were assessed through the utilization of multiple bioinformatics tools including PROVEAN, SNP&GO, SIFT, and PolyPhen2. Structural and functional effects of these variants on protein structures were evaluated through the use of additional tools such as I-Mutant, MutPred, and ConSurf. Three-dimensional (3D) models of both the wild-type and mutant proteins were constructed through the utilization of I-TASSER software, with subsequent structural comparisons between the models conducted through the use of the TM-align score. A total of 500 individuals, 250 cases and 250 controls, were genotyped through Tri-ARMS-PCR method and the resultant data was statistically analyzed using various inheritance models. Our bioinformatics analysis showed significant structural differences for wild type and mutant protein (TM-scores and RMSD values were 0.85934 and 2.34 for rs2397084 (E126G), 0.87388 and 2.49 for rs11465553 (V155I), and 0.86572 and 0.86572 for rs763780 (H161R) with decrease stability for the later. Overall, these tools enabled us to predict that these variants are crucial in causing disease phenotypes. We further tested each of these single nucleotide variants for their association with RA. Our analysis revealed a strong positive association between the genetic variant rs763780 and the risk of developing rheumatoid arthritis (RA) at both the genotypic and allelic levels. The genotypic association was statistically significant[χ2 = 111.8; P value <0.0001], as was the allelic level [OR 3.444 (2.539-4.672); P value 0.0008]. These findings suggest that the presence of this genetic variant may increase the susceptibility to RA. Similarly, we observed a significant distribution of the genetic variant rs11465553 at the genotypic level [χ2 = 25.24; P value = 0.0001]. However, this variant did not show a significant association with RA at the allelic level [OR = 1.194 (0.930-1.531); P value = 0.183]. However, the distribution of variant rs2397084 was more or less random across our sample with no significant association either at genotypic and or allelic level. Put together, our association study and in silico prediction of decreasing of IL17-F protein stabilty confirmed that two SNPs, rs11465553 and rs763780 are crucial to the suscetibility of and showed that these RA in Pakistani patients.

Early whole-body mutant huntingtin lowering averts changes in proteins and lipids important for synapse function and white matter maintenance in the LacQ140 mouse model.

Expansion of a triplet repeat tract in exon 1 of the HTT gene causes Huntington's disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. Lowering mHTT is a promising approach to treat HD, but it is unclear when lowering should be initiated, how much is necessary, and what duration should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. Using a mHtt-inducible mouse model, we analyzed mHtt lowering initiated at different ages and sustained for different time-periods. mHTT protein in cytoplasmic and synaptic compartments of the striatum was reduced 38-52%; however, there was minimal lowering of mHTT in nuclear and perinuclear regions where aggregates formed at 12 months of age. Total striatal lipids were reduced in 9-month-old LacQ140 mice and preserved by mHtt lowering. Subclasses important for white matter structure and function including ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (MGDG), contributed to the reduction in total lipids. Phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA) were also changed in LacQ140 mice. Levels of all subclasses except ceramide were preserved by mHtt lowering. mRNA expression profiling indicated that a transcriptional mechanism contributes to changes in myelin lipids, and some but not all changes can be prevented by mHtt lowering. Our findings suggest that early and sustained reduction in mHtt can prevent changes in levels of select striatal proteins and most lipids, but a misfolded, degradation-resistant form of mHTT hampers some benefits in the long term.

Predicting pathogenic protein variants.

Machine-learning algorithm uses structure prediction to spot disease-causing mutations.

K-Pro: Kinetics Data on Proteins and Mutants.

The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's ß and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers.

Pathogenic Aß production by heterozygous PSEN1 mutations is intrinsic to the mutant protein and not mediated by conformational hindrance of wild-type PSEN1.

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer's disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-ß peptides (Aß42 and/or Aß43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aß production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aß42/Aß40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aß42/Aß40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aß production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.

Discovery of the allosteric inhibitor from actinomyces metabolites to target EGFRCSTMLR mutant protein: molecular modeling and free energy approach.

EGFR (epidermal growth factor receptor), a surface protein on the cell, belongs to the tyrosine kinase family, responsible for cell growth and proliferation. Overexpression or mutation in the EGFR gene leads to various types of cancer, i.e., non-small cell lung cancer, breast, and pancreatic cancer. Bioactive molecules identified in this genre were also an essential source of encouragement for researchers who accomplished the design and synthesis of novel compounds with anticancer properties. World Health Organization (WHO) report states that antibiotic resistance is one of the most severe risks to global well-being, food safety, and development. The world needs to take steps to lessen this danger, such as developing new antibiotics and regulating their use. In this study, 6524 compounds derived from Streptomyces sp. were subjected to drug-likeness filters, molecular docking, and molecular dynamic simulation for 1000 ns to find new triple mutant EGFRCSTMLR (EGFR-L858R/T790M/C797S) inhibitors. Docking outcomes revealed that five compounds showed better binding affinity (- 9.074 to - 9.3 kcal/mol) than both reference drug CH7233163 (- 6.11 kcal/mol) and co-crystallized ligand Osimertinib (- 8.07 kcal/mol). Further, molecular dynamic simulation confirmed that ligand C_42 exhibited the best interaction at the active site of EGFR protein and comprised a better average radius of gyration (3.87 Å) and average SASA (Solvent Accessible Surface Area) (82.91 Å2) value than co-crystallized ligand (4.49 Å, 222.38 Å2). Additionally, its average RMSD (Root Mean Square Deviation) (3.25 Å) and RMSF (Root Mean Square Fluctuation) (1.54 Å) values were highly similar to co-crystallized ligand (3.07 Å, 1.54 Å). Compared to the reference ligand, it also demonstrated conserved H-bond interactions with the residues MET_793 and GLN_791 with strong interaction probability. In conclusion, we have found a potential drug with no violation of the rule of three, Lipinski's rule of five, and 26 other vital parameters having great potential in medicinal and pharmaceutical industries applications and can overcome synthetic drug issues.

Extension of the DNAJB2a isoform in a dominant neuromyopathy family.

Recessive mutations in the DNAJB2 gene, encoding the J-domain co-chaperones DNAJB2a and DNAJB2b, have previously been reported as the genetic cause of progressive peripheral neuropathies, rarely involving pyramidal signs, parkinsonism and myopathy. We describe here a family with the first dominantly acting DNAJB2 mutation resulting in a late-onset neuromyopathy phenotype. The c.832 T > G p.(*278Glyext*83) mutation abolishes the stop codon of the DNAJB2a isoform resulting in a C-terminal extension of the protein, with no direct effect predicted on the DNAJB2b isoform of the protein. Analysis of the muscle biopsy showed reduction of both protein isoforms. In functional studies, the mutant protein mislocalized to the endoplasmic reticulum due to a transmembrane helix in the C-terminal extension. The mutant protein underwent rapid proteasomal degradation and also increased the turnover of co-expressed wild-type DNAJB2a, potentially explaining the reduced protein amount in the patient muscle tissue. In line with this dominant negative effect, both wild-type and mutant DNAJB2a were shown to form polydisperse oligomers.

Precise pancreatic cancer therapy through targeted degradation of mutant p53 protein by cerium oxide nanoparticles.

BACKGROUND: In a significant proportion of cancers, point mutations of TP53 gene occur within the DNA-binding domain, resulting in an abundance of mutant p53 proteins (mutp53) within cells, which possess tumor-promoting properties. A potential and straightforward strategy for addressing p53-mutated cancer involves the induction of autophagy or proteasomal degradation. Based on the previously reported findings, elevating oxidative state in the mutp53 cells represented a feasible approach for targeting mutp53. However, the nanoparticles previous reported lacked sufficient specificity of regulating ROS in tumor cells, consequently resulted in unfavorable toxicity in healthy cells. RESULTS: We here in showed that cerium oxide CeO2 nanoparticles (CeO2 NPs) exhibited an remarkable elevated level of ROS production in tumor cells, as compared to healthy cells, demonstrating that the unique property of CeO2 NPs in cancer cells provided a feasible solution to mutp53 degradation. CeO2 NPs elicited K48 ubiquitination-dependent degradation of wide-spectrum mutp53 proteins in a manner that was dependent on both the dissociation of mutp53 from the heat shock proteins Hsp90/70 and the increasing production of ROS. As expected, degradation of mutp53 by CeO2 NPs abrogated mutp53-manifested gain-of-function (GOF), leading to a reduction in cell proliferation and migration, and dramatically improved the therapeutic efficacy in a BxPC-3 mutp53 tumor model. CONCLUSIONS: Overall, CeO2 NPs increasing ROS specifically in the mutp53 cancer cells displayed a specific therapeutic efficacy in mutp53 cancer and offered an effective solution to address the challenges posed by mutp53 degradation, as demonstrated in our present study.

Global Analysis of Multi-Mutants to Improve Protein Function.

The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to a previously published experiment reporting on >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions (Sarkisyan et al., 2016). The GMMA method achieves a good fit to this dataset while being analytically transparent. We show experimentally that the six top-ranking substitutions progressively enhance GFP. More broadly, using only a single experiment as input our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function. In conclusion, we suggest that large libraries of multiply-substituted variants may provide a unique source of information for protein engineering.

Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject.

Familial hypercholesterolaemia (FH) is an autosomal dominant dyslipidaemia, characterised by elevated LDL cholesterol (LDL-C) levels in the blood. Three main genes are involved in FH diagnosis: LDL receptor (LDLr), Apolipoprotein B (APOB) and Protein convertase subtilisin/kexin type 9 (PCSK9) with genetic mutations that led to reduced plasma LDL-C clearance. To date, several PCSK9 gain-of-function (GOF) variants causing FH have been described based on their increased ability to degrade LDLr. On the other hand, mutations that reduce the activity of PCSK9 on LDLr degradation have been described as loss-of-function (LOF) variants. It is therefore important to functionally characterise PCSK9 variants in order to support the genetic diagnosis of FH. The aim of this work is to functionally characterise the p.(Arg160Gln) PCSK9 variant found in a subject suspected to have FH. Different techniques have been combined to determine efficiency of the autocatalytic cleavage, protein expression, effect of the variant on LDLr activity and affinity of the PCSK9 variant for the LDLr. Expression and processing of the p.(Arg160Gln) variant had a result similar to that of WT PCSK9. The effect of p.(Arg160Gln) PCSK9 on LDLr activity is lower than WT PCSK9, with higher values of LDL internalisation (13%) and p.(Arg160Gln) PCSK9 affinity for the LDLr is lower than WT, EC50 8.6 ± 0.8 and 25.9 ± 0.7, respectively. The p.(Arg160Gln) PCSK9 variant is a LOF PCSK9 whose loss of activity is caused by a displacement of the PCSK9 P' helix, which reduces the stability of the LDLr-PCSK9 complex.

Biochemical characterization of two novel mutations in the human high-affinity choline transporter 1 identified in a patient with congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high-affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to reuptake choline. Biallelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, which we identified in an 11-year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life.

Bioinformatics Insights on the Physicochemical Properties of SCN5A Mutant Proteins Associated with the Brugada Syndrome.

BACKGROUND: The Brugada syndrome (BrS) is a heart rhythm condition that is commonly associated with a strong predisposition for sudden cardiac death. Malignant ventricular arrhythmias could occur secondary to the dysfunction of the cardiac sodium voltage-gated Na(v)1.5 channel (SCN5A). OBJECTIVE: This study aimed to perform a multiparametric computational analysis of the physicochemical properties of SCN5A mutants associated with BrS using a set of bioinformatics tools. METHODS: In-house algorithms were calibrated to calculate, in a double-blind test, the Polarity Index Method (PIM) profile and protein intrinsic disorder predisposition (PIDP) profile of each sequence, and computer programs specialized in the genomic analysis were used. RESULTS: Specific regularities in the charge/polarity and PIDP profile of the SCN5A mutant proteins enabled the re-creation of the taxonomy, allowing us to propose a bioinformatics method that takes advantage of the PIM profile to identify this group of proteins from their sequence. CONCLUSION: Bioinformatics programs could reproduce characteristic PIM and PIDP profiles of the BrS-related SCN5A mutant proteins. This information can contribute to a better understanding of these altered proteins.