Reduced Protein Stability Research Articles

Freeze-drying of HESylated IFNα-2b: Effect of HESylation on storage stability in comparison to PEGylation

A comparison of lyophilized PEGylated and HESylated IFNα was carried out to investigate the influence of protein conjugation, lyoprotectants as well as storage temperature on protein stability. Results show that PEG tends to crystallize during freeze-drying, reducing protein stability upon storage. In contrast, HESylation® drastically improved the stability over PEGylation by remaining totally amorphous during lyophilization, with and without lyoprotectants while providing a high glass transition temperature of the freeze-dried cakes.

Mistranslation drives the evolution of robustness in TEM-1 β-lactamase

How biological systems such as proteins achieve robustness to ubiquitous perturbations is a fundamental biological question. Such perturbations include errors that introduce phenotypic mutations into nascent proteins during the translation of mRNA. These errors are remarkably frequent. They are also costly, because they reduce protein stability and help create toxic misfolded proteins. Adaptive evolution might reduce these costs of protein mistranslation by two principal mechanisms. The first increases the accuracy of translation via synonymous "high fidelity" codons at especially sensitive sites. The second increases the robustness of proteins to phenotypic errors via amino acids that increase protein stability. To study how these mechanisms are exploited by populations evolving in the laboratory, we evolved the antibiotic resistance gene TEM-1 in Escherichia coli hosts with either normal or high rates of mistranslation. We analyzed TEM-1 populations that evolved under relaxed and stringent selection for antibiotic resistance by single molecule real-time sequencing. Under relaxed selection, mistranslating populations reduce mistranslation costs by reducing TEM-1 expression. Under stringent selection, they efficiently purge destabilizing amino acid changes. More importantly, they accumulate stabilizing amino acid changes rather than synonymous changes that increase translational accuracy. In the large populations we study, and on short evolutionary timescales, the path of least resistance in TEM-1 evolution consists of reducing the consequences of translation errors rather than the errors themselves.

Spectroscopy and computational studies on the interaction of octyl, dodecyl, and hexadecyl derivatives of anionic and cationic surfactants with adenosine deaminase

Effects of sodium (octyl, dodecyl, hexadecyl) sulfate and their cationic analogous on the structure of adenosine deaminase (ADA) were investigated by fluorescence and circular dichroism spectroscopy as well as molecular dynamics simulation and docking calculation. Root-mean-square derivations, radius of gyration, solvent accessible surface area, and radial distribution function were obtained. The results showed that anionic and cationic surfactants reduce protein stability. Cationic surfactants have more effect on the ADA structure in comparison with anionic surfactants. More concentration and longer surfactants are parallel to higher denaturation. Furthermore, aggregation in the presence of anionic surfactants is more than cationic surfactants. Docking data showed that longer surfactants have more interaction energy and smaller ones bound to the active site.

Mutations in DCHS1 cause mitral valve prolapse.

SUMMARYMitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals1–3. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery4,5. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds) that segregates with MVP in the family. Morpholino knockdown of the zebrafish homolog dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 mRNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells, and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs as well as in Dchs1+/− mouse MVICs result in altered migration and cellular patterning, supporting these processes as etiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Abstract 2601: A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer in vitro and in vivo

Abstract For decades, lung cancer has been a leading cause of death in the U.S., mainly due to oncogenic signaling pathways activated by carcinogens such as smoking. Various carcinogens induce EGFR/RAS/MAPK signaling that is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 against lung carcinogenesis will be beneficial to these patients. Herein, we examined the therapeutic effects of catechol (also known as pyrocatechol or 1,2-dihydroxybenzene), a naturally occurring chemical in many foods such as apple, banana, apricot, grape, plum, avocado and mushroom. Co-crystallography results suggested that ERK2 is a direct target of catechol. In agreement with the co-crystallography result, catechol was confirmed to inhibit ERK2 kinase activity in vitro. Catechol inhibited anchorage-independent KP2 and H460 lung cancer cell growth in a dose-dependent manner. Phosphorylation of c-Myc, a substrate of ERK2, was reduced in catechol-treated lung cancer cells compared to untreated controls. Decreased c-Myc phosphorylation resulted in its reduced protein stability and subsequent down-regulation of total c-Myc levels. Treatment with catechol induced G1 cell cycle arrest and resulted in a decrease of G1 phase related proteins, CDK2 and cyclin D1. In vivo study results also revealed that catechol inhibited growth of KP2 and H460 xenograft tumors. Phosphorylation levels of c-Myc were also strongly decreased by catechol treatment in vivo. Taken together, we have shown that catechol exerts inhibitory effects on the ERK2/c-Myc signaling axis in lung cancer cells to reduce tumor growth in vitro and in vivo. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis. Citation Format: Do Young Lim, Seung Ho Shin, Margarita Malakhova, Mee-Hyun Lee, Ann M. Bode, Zigang Dong. A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2601. doi:10.1158/1538-7445.AM2015-2601

Abstract 4790: Noncanonical functions of EZH2 in triple-negative breast cancers

Abstract Background: Triple negative (estrogen receptor, progesterone receptor, HER2-neu negative) breast cancers (TNBC) account for 15% of all breast cancers but are responsible for a disproportionate number of deaths. Overexpression of the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is an independent prognostic biomarker significantly associated with poorly-differentiated TNBCs and poor patient outcome. As a member of the polycomb repressive complex 2 (PRC2), the canonical function of EZH2 is to mediate transcriptional repression of target genes via trimethylation of histone H3 at lysine 27 (H3K27me3). We previously identified a novel interaction between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of progression and metastasis of TNBC. We found that EZH2 can bind to p38 and that overexpression of EZH2 leads to phosphorylation and activation of p38. Based on these data and preliminary in vitro studies, we hypothesized that EZH2 may have functions independent of its H3K27me3 activity in TNBC, where it can methylate p38 and other non-histone proteins. We further hypothesized that these methylation events may be important for p38 activation or stability. Methods: In order to test this hypothesis, we performed an in vitro methyltransferase assay with recombinant PRC2 complex, using p38α as substrate. We then employed LC MS/MS to determine the lysine residues methylated on p38α by PRC2. In order to determine the functional consequences of this modification, we assessed for changes in p38 isoform protein stability in a cycloheximide pulse-chase assay with genetic or pharmacologic inhibition of EZH2 in MDA-MB-231 and SUM149 triple-negative breast cancer cell lines. As a complementary approach, we used site-directed mutagenesis to block methylation at these sites and to determine resulting changes in protein stability. Results: EZH2 methylates p38α in vitro at two lysine residues. Enzymatic inhibition of EZH2 results in reduced levels of methylated p38α, as well as decreased stability in vivo in an isoform-specific manner. Consistent with these results, lysine-to-alanine mutagenesis of these residues results in significantly reduced protein stability. We are currently investigating the possibility that EZH2 methylation may block other modifications, such as ubiquitination, at these sites. Conclusions: We provide evidence that EZH2 directly methylates oncoprotein p38α at two novel sites and that these modifications confer stability. Taken together, our data suggest that p38 methylation may represent an additional mechanism by which EZH2 contributes to TNBC progression. Citation Format: Talha Anwar, Heather Moore, Sarah Bergholtz, Celina Kleer. Noncanonical functions of EZH2 in triple-negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4790. doi:10.1158/1538-7445.AM2015-4790

14-3-3 Binding and Sumoylation Concur to the Down-Modulation of β-catenin Antagonist chibby 1 in Chronic Myeloid Leukemia.

The down-modulation of the β-catenin antagonist Chibby 1 (CBY1) associated with the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of β-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

Computational study of missense mutations in phenylalanine hydroxylase.

Hyperphenylalaninemia (HPA) is one of the most common metabolic disorders. HPA, which is transmitted by an autosomal recessive mode of inheritance, is caused by mutations of the phenylalanine hydroxylase gene. Most mutations are missense and lead to reduced protein stability and/or impaired catalytic function. The impact of such mutations varies, ranging from classical phenylketonuria (PKU), mild PKU, to non-PKU HPA phenotypes. Despite the fact that HPA is a monogenic disease, clinical data show that one PKU genotype can be associated with more in vivo phenotypes, which indicates the role of other (still unknown) factors. To better understand the phenotype-genotype relationships, we analyzed computationally the impact of missense mutations in homozygotes stored in the BIOPKU database. A total of 34 selected homozygous genotypes was divided into two main groups according to their phenotypes: (A) genotypes leading to non-PKU HPA or combined phenotype non-PKU HPA/mild PKU and (B) genotypes leading to classical PKU, mild PKU or combined phenotype mild PKU/classical PKU. Combining in silico analysis and molecular dynamics simulations (in total 3μs) we described the structural impact of the mutations, which allowed us to separate 32 out of 34 mutations between groups A and B. Testing the simulation conditions revealed that the outcome of mutant simulations can be modulated by the ionic strength. We also employed programs SNPs3D, Polyphen-2, and SIFT but based on the predictions performed we were not able to discriminate mutations with mild and severe PKU phenotypes.

No contribution of the ABCB11 p.444A polymorphism in Japanese patients with drug-induced cholestasis.

European studies have revealed that the ABCB11 c.1331T>C (V444A) polymorphism (rs2287622) C-allele frequency is higher among patients with drug-induced cholestasis. Given the low incidence of this disease, however, this association has not been sufficiently elucidated. We aimed to investigate the significance of this polymorphism in Japanese patients. We determined ABCB11 V444A polymorphism frequencies and HLA genotypes in two independent drug-induced cholestasis cohorts. Expression and taurocholate transport activity of proteins from 444A variants were analyzed using Madin-Darby canine kidney II cells. In cohort 1 (n = 40), the V444A polymorphism C-allele frequency (66%) was lower than that in controls (n = 190, 78%), but this difference was not significant (P = 0.09). In cohort 2 (n = 119), comprising patients with cholestatic (n = 19), hepatocellular (n = 74), and mixed (n = 26) liver injuries, the C-allele frequency was lower among patients with cholestatic liver injury (68%) than among those with hepatocellular (75%) or mixed liver injury (83%), although this difference was not significant. In cohort 1, HLA-A*0201 was observed more frequently in patients (22%) than in controls [11%; P = 0.003; odds ratio, 2.4 (95% confidence interval, 1.4-4.0)]. Taurocholate transport activity of 444A-encoded protein was significantly lower than that of 444V-encoded protein (81% of 444V, P < 0.05) because of the reduced protein stability. In conclusion, ABCB11 444A had slightly reduced transport activity, but it did not contribute to the occurrence of drug-induced cholestasis in Japanese patients. Therefore, genetic susceptibility to acquired cholestasis may differ considerably by ethnicity.

Mutations in the intellectual disability gene KDM5C reduce protein stability and demethylase activity.

Mutations in KDM5C are an important cause of X-linked intellectual disability in males. KDM5C encodes a histone demethylase, suggesting that alterations in chromatin landscape may contribute to disease. We used primary patient cells and biochemical approaches to investigate the effects of patient mutations on KDM5C expression, stability and catalytic activity. We report and characterize a novel nonsense mutation, c.3223delG (p.V1075Yfs*2), which leads to loss of KDM5C protein. We also characterize two KDM5C missense mutations, c.1439C>T (p.P480L) and c.1204G>T (p.D402Y) that are compatible with protein production, but compromise stability and enzymatic activity. Finally, we demonstrate that a c.2T>C mutation in the translation initiation codon of KDM5C results in translation re-start and production of a N-terminally truncated protein (p.M1_E165del) that is unstable and lacks detectable demethylase activity. Patient fibroblasts do not show global changes in histone methylation but we identify several up-regulated genes, suggesting local changes in chromatin conformation and gene expression. This thorough examination of KDM5C patient mutations demonstrates the utility of examining the molecular consequences of patient mutations on several levels, ranging from enzyme production to catalytic activity, when assessing the functional outcomes of intellectual disability mutations.

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

Background:LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein.Methods:Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5.Results:Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network.Conclusions:Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

Abstract 18977: Non-Syndromic Mitral Valve Prolapse From Gene Mutations to Modifiable Mechanisms

Mitral valve prolapse (MVP) is a common disease affecting nearly 1 in 40 individuals. It is the leading surgical indication for mitral valve regurgitation. Despite a clear heritable component, the genetic etiology of non-syndromic MVP has remained elusive. Individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of a previously linked interval on chromosome 11. We found a novel missense mutation in DCHS1, the human homologue of the Drosophila cell polarity gene dachsous (ds) that segregates with MVP in the family. The familial mutation reduced protein stability in zebrafish and cultured cells. Morpholino knockdown of zebrafish dachsous1b yielded a regurgitant atrioventricular canal defect that could be rescued by wild-type human DCHS1 but not by mRNA with the familial mutation. Adult haploinsufficient Dchs1 mice had anatomic and functional prolapse of thickened, elongated leaflets recapitulating the human MVP phenotype. Dchs1 haploinsufficiency was associated with developmental defects in valve shape, cell migration and patterning during fetal morphogenesis, suggesting cell polarity as a previously unrecognized facet of valve morphogenesis. Studies of downstream changes identified increased MAP kinase activities and α-SMA+ cells (myofibroblasts) in the valves preceding myxomatous degeneration; these suggest potential future directions for blunting disease progression. Additional genetic studies identified a second family with a DCHS1 mutation and a significantly increased burden of novel DCHS1 mutations in sporadic MVP cases Interdisciplinary studies strongly support DCHS1 as the first identified disease-causing gene for non-syndromic MVP and suggest potential mechanisms for modifying disease natural history.

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity

BackgroundDilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. MethodsForce of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed. ResultsA decrease of −0.26 and −0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50=5.61) compared to control cTnI-WT.cTnC (pCa50=5.75), similarly affecting cTnT-ΔE96 (pCa50=6.03) compared to control \\cTnI-WT.cTnC (pCa50=6.14). Fluorescence studies measuring cTnCIAANS Ca2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT. ConclusionsAlthough ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions. General significanceThe pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.

TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells.

High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma.

Ligand-binding pocket bridges DNA-binding and dimerization domains of the urate-responsive MarR homologue MftR from Burkholderia thailandensis.

Members of the multiple antibiotic resistance regulator (MarR) family often regulate gene activity by responding to a specific ligand. In the absence of ligand, most MarR proteins function as repressors, while ligand binding causes attenuated DNA binding and therefore increased gene expression. Previously, we have shown that urate is a ligand for MftR (major facilitator transport regulator), which is encoded by the soil bacterium Burkholderia thailandensis. We show here that both mftR and the divergently oriented gene mftP encoding a major facilitator transport protein are upregulated in the presence of urate. MftR binds two cognate sites in the mftR-mftP intergenic region with equivalent affinity and sensitivity to urate. Mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter PecS significantly reduced protein stability and DNA binding affinity but not ligand binding. These data suggest that residues equivalent to those implicated in ligand binding to HucR and PecS serve structural roles and that MftR relies on distinct residues for ligand binding. MftR exhibits a two-step melting transition suggesting independent unfolding of the dimerization and DNA-binding regions; urate binding or mutations in the predicted ligand-binding sites result in one-step unfolding transitions. We suggest that MftR binds the ligand in a cleft between the DNA-binding lobes and the dimer interface but that the mechanism of ligand-mediated attenuation of DNA binding differs from that proposed for other urate-responsive MarR homologues. Since DNA binding by MftR is attenuated at 37 °C, our data also suggest that MftR responds to both ligand and a thermal upshift by attenuated DNA binding and upregulation of the genes under its control.

SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner.

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD+-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis.

Improvements in Algorithms for Phenotype Inference: The NAT2 Example

Numerous studies have analyzed the impact of N-acetyltransferase 2 (NAT2) polymorphisms on drug efficacy, side effects as well as cancer risk. Here, we present the state of the art of deriving haplotypes from polymorphisms and discuss the available software. PHASE v2.1 is currently considered a gold standard for NAT2 haplotype assignment. In vitro studies have shown that some slow acetylation genotypes confer reduced protein stability. This has been observed particularly for G191A, T341C and G590A. Substantial ethnic variations of the acetylation status have been described. Probably, upcoming agriculture and the resulting change in diet caused a selection pressure for slow acetylation. In recent years much research has been done to reduce the complexity of NAT2 genotyping. Deriving the haplotype from seven SNPs is still considered a gold standard. However, meanwhile several studies have shown that a two-SNP combination, C282T and T341C, results in a similarly good distinction in Caucasians. However, attempts to further reduce complexity to only one 'tagging SNP' (rs1495741) may lead to wrong predictions where phenotypically slow acetylators were genotyped as intermediate or rapid. Numerous studies have shown that slow NAT2 haplotypes are associated with increased urinary bladder cancer risk and increased risk of anti-tuberculosis drug-induced hepatotoxicity. A drawback of the current practice of solely discriminating slow, intermediate and rapid genotypes for phenotype inference is limited resolution of differences between slow acetylators. Future developments to differentiate between slow and ultra-slow genotypes may further improve individualized drug dosing and epidemiological studies of cancer risk.

Loss of ALDH18A1 function is associated with a cellular lipid droplet phenotype suggesting a link between autosomal recessive cutis laxa type 3A and Warburg Micro syndrome.

Autosomal recessive cutis laxa type 3A is caused by mutations in ALDH18A1, a gene encoding the mitochondrial enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS). It is a rare disorder with only six pathogenic mutations and 10 affected individuals from five families previously described in the literature. Here we report the identification of novel compound heterozygous missense mutations in two affected siblings from a Lebanese family by whole-exome sequencing. The mutations alter a conserved C-terminal domain of the encoded protein and reduce protein stability as determined through Western blot analysis of patient fibroblasts. Patient fibroblasts exhibit a lipid droplet phenotype similar to that recently reported in Warburg Micro syndrome, a disorder with similar features but hitherto unrelated cellular etiology.

A novel Zap70 mutation with reduced protein stability demonstrates the rate‐limiting threshold for Zap70 in T‐cell receptor signalling

Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.

The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase.

Mutation of ABO glycosyltransferase (GT) can cause protein stability changes that can result in a weak ABO phenotype. To explain the Bw phenotype of a novel ABO*Bw allele, a protein stability of the mutant GT, which enhances the information of the three-dimensional (3D) structural analysis, was calculated. ABO serology and genotyping were performed on a neonate and her five family members. A 3D structural analysis of the wild-type GTB and enzymes with a variety of mutations at Residue 168, along with predicted protein stability changes (ΔΔG) and flow cytometric analysis of ABO antigen expression on HeLa cells transfected with plasmids containing R168Q, R168L, and R168P mutants was also performed. A novel ABO*Bw allele (c.503G>A, p.R168Q) was discovered. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTB, and the ΔΔG values, which inversely correlated with the mean relative fluorescence intensity of ABO antigen expression, quantitatively explained the reduced ABO antigen expression. The predicted protein stability change of a mutant GT enzyme might be a useful and convenient approach to objectively and quantitatively explain the reduced ABO antigen expression.