Ser Mutation Research Articles

Expression of sensitized β2 nAChR subunits in VTA neurons enhances intravenous nicotine self-administration in male rats

Ventral tegmental area (VTA) nicotinic acetylcholine receptors (nAChRs) are important for nicotine reinforcement. To determine whether and to what extent these receptors are sufficient for nicotine reinforcement, we expressed β2Leu9′Ser (i.e. sensitized) nAChR subunits in the VTA of adult male rats and assessed the nicotine dose-response relationship in intravenous self-administration (SA). β2Leu9′Ser rats self-administered nicotine doses 50–100 fold lower than the lowest doses that control rats would respond for. Expression of WT β2 subunits confirmed that this enhanced sensitivity to nicotine was due to the Leu9′Ser mutation in β2. Higher unit doses were associated with strong escalation in β2Leu9′Ser rats over 17 fixed ratio sessions. Escalation was minimal or absent in control rats at the same unit doses. In progressive ratio SA, β2Leu9′Ser rats exhibited higher breakpoints than control rats when the nicotine unit dose was 1.5 μg/kg/inf or higher. In intermittent access SA, β2Leu9′Ser rats exhibited response patterns very similar to control rats. By adding nicotine dose-response data, progressive ratio assays, and intermittent access results that rule out stereotypy, these data significantly extend our previous finding that nicotine activation of the mesolimbic dopamine pathway is sufficient for nicotine reinforcement.

The mutation of Japanese encephalitis virus envelope protein residue 389 attenuates viral neuroinvasiveness

The envelope (E) protein of the Japanese encephalitis virus (JEV) is a key protein for virus infection and adsorption of host cells, which determines the virulence of the virus and regulates the intensity of inflammatory response. The mutation of multiple aa residues in the E protein plays a critical role in the attenuated strain of JEV. This study demonstrated that the Asp to Gly, Ser, and His mutation of the E389 site, respectively, the replication ability of the viruses in cells was significantly reduced, and the viral neuroinvasiveness was attenuated to different degrees. Among them, the mutation at E389 site enhanced the E protein flexibility contributed to the attenuation of neuroinvasiveness. In contrast, less flexibility of E protein enhanced the neuroinvasiveness of the strain. Our results indicate that the mechanism of attenuation of E389 aa mutation attenuates neuroinvasiveness is related to increased flexibility of the E protein. In addition, the increased flexibility of E protein enhanced the viral sensitivity to heparin inhibition in vitro, which may lead to a decrease in the viral load entering brain. These results suggest that E389 residue is a potential site affecting JEV virulence, and the flexibility of the E protein of aa at this site plays an important role in the determination of neuroinvasiveness.

Glutathione transferase P1 is modified by palmitate.

Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7

The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.

Effect of alanine versus serine at position 88 of human transthyretin mutants on the protein stability.

Human transthyretin (TTR) is a homo-tetrameric plasma protein associated with a high percentage of β-sheet forming amyloid fibrils. It accumulates in tissues or extracellular matrices to cause amyloid diseases. Free energy simulations with thermodynamic integration based on all-atom molecular dynamics simulations have been carried out to analyze the effects of the His88→Ala and Ser mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His88→Ala and His88→Ser mutations are -1.84±0.86 and 7.56±0.55kcal/mol, respectively, which are in excellent agreement with prior reported experimental values. The simulation results show that the H88A mutant is more stable than the wild type, whereas the H88S mutant is less stable than the wild type. The free energy component analysis shows that the contribution to the free energy change difference (ΔΔG) for the His88→Ala and His88→Ser mutations mainly arise from electrostatic and van der Waals interactions, respectively. The electrostatic term stabilizes the H88A mutant more than the wild type, but the van der Waals interaction destabilizes the H88S mutant relative to the wild type. Individual residue contributions to the free energy change show neighboring residues exert stabilizing and destabilizing influence on the mutants. The implications of the simulation results for understanding the stabilizing and destabilizing effect and its contribution to protein stability are discussed.

Novel mutation in SMPD1 gene found by whole-exome sequencing in Niemann-Pick disease patient

Niemann-Pick diseases (NPD) are heterogeneous and autosomal recessive disorders and are classified into four types: type A, type B, type C1, and type C2 that type A and B Niemann–Pick is induced via SMPD1 gene mutations. Accumulation of sphingomyelin in the central nervous system is the first and prominent cause of the classical infantile type (type A) of NPD which is the most extreme form of NPD and causes severe central nervous system decay, hepatosplenomegaly, and fatal disorder. Our aim with this study was to propose that the novel likely pathogenic variant, c.1586G>C (p.529Trp>Ser) can cause Niemann-Pick disease type A. Present study involves a 2 years-old child as a result of a consanguineous marriage that has been diagnosed with clumsiness and gait disturbance at the beginning, and include hypotonia, microcephaly, severe hepatosplenomegaly in infancy, speech and developmental delay, dysphagia during the disease course. Considering the symptoms of Niemann-Pick disease, the patient's DNA was extracted and used for molecular studying and sequencing. Performing WES on proband identified a novel likely pathogenic, c.1586G>C (p.529Trp>Ser) mutation of the SMPD1 gene. This gene has an autosomal recessive inheritance pattern. To date, c.1586G>C (p.529Trp>Ser) likely pathogenic variant has not been observed or reported in the world. This mutation locates in the 6415659 positions and disrupts the function of the SMPD1 gene, causing symptoms of Niemann-Pick disease type A. The mutation reported in this article is associated with the NPD type A, disease phenotype, molecular findings and clinical observations underscore it.

Cysteine Oxidation to Sulfenic Acid in APE1 Aids G-Quadruplex Binding While Compromising DNA Repair.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a base excision repair (BER) enzyme that is also engaged in transcriptional regulation. Previous work demonstrated that the enzymatic stalling of APE1 on a promoter G-quadruplex (G4) recruits transcription factors during oxidative stress for gene regulation. Also, during oxidative stress, cysteine (Cys) oxidation is a post-translational modification (PTM) that can change a protein's function. The current study provides a quantitative survey of cysteine oxidation to sulfenic acid in APE1 and how this PTM at specific cysteine residues affects the function of APE1 toward the NEIL3 gene promoter G4 bearing an abasic site. Of the seven cysteine residues in APE1, five (C65, C93, C208, C296, and C310) were prone to carbonate radical anion oxidation to yield sulfenic acids that were identified and quantified by mass spectrometry. Accordingly, five Cys-to-serine (Ser) mutants of APE1 were prepared and found to have attenuated levels of endonuclease activity, depending on the position, while KD values generally decreased for G4 binding, indicating greater affinity. These data support the concept that cysteine oxidation to sulfenic acid can result in modified APE1 that enhances G4 binding at the expense of endonuclease activity during oxidative stress. Cysteine oxidation to sulfenic acid residues should be considered as one of the factors that may trigger a switch from base excision repair activity to transcriptional modulation by APE1.

Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095

The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

Behavioral and neurological analyses of adult mice carrying null and distinct loss-of-receptor function mutations in protein tyrosine phosphatase receptor type Z (PTPRZ).

Protein tyrosine phosphatase receptor type Z (PTPRZ) is preferentially expressed in the central nervous system as two transmembrane receptor isoforms PTPRZ-A/B and one secretory isoform PTPRZ-S. Ptprz-knockout mice lacking the expression of all three isoforms show behavioral, learning, and neurological abnormalities, including increased exploratory activities to novelty, deficits in spatial and contextual learning, and reduced responses to methamphetamine, relative to wild-type mice. To investigate whether PTPRZ isoforms play distinct physiological roles, we herein performed behavioral studies on two knock-in mouse lines: One expresses the catalytically inactive Cys-1930 to Ser (CS) mutants of PTPRZ-A/B, while the other generated in the present study expresses catalytically active mutants of PTPRZ-A/B lacking the negative regulatory PTP-D2 domain and C-terminal PDZ-binding motif (ΔD2) instead of wild-type PTPRZ-A/-B. In contrast to Ptprz-knockout mice, neither increased responses to novelty in the open field nor memory impairments in the inhibitory-avoidance task were observed in Ptprz-CS or Ptprz-ΔD2 mice. However, the effects of methamphetamine on locomotor activity were significantly weaker in Ptprz-KO mice and CS mutant mice than in wild-type mice, but were normal in ΔD2 mutant mice. Furthermore, microdialysis experiments revealed that methamphetamine-evoked dopamine release in the nucleus accumbens was reduced in Ptprz-KO mice and CS mutant mice. These results suggest that the extracellular region of PTPRZ, including the secretory isoform, is crucial for behavioral responses to novelty and the formation of aversive memories, whereas the PTPase activities of PTPRZ receptor isoforms are involved in regulating the dopaminergic system.

WNT10A rs147680216 G>A mutation indicates a higher risk for non-syndromic oral cleft in a northeastern Chinese population

Non-syndromic oral cleft lip and palate is a heterogeneous group of congenital malformations that consist of cleft palate, and cleft lip with or without cleft palate. The members of the wingless type mouse mammary tumour virus (MMTV) integration site family (Wnts) regulate various developmental processes including craniofacial development, and have a role in that of cleft lip and palate. We aimed to identify the potential polymorphisms in the Wnt10a gene, and to explore the association between the variations in the gene and the risk of development of cleft palate. A total of 198 affected patients (cleft lip, n=67; cleft palate, n=48; and both, n=83) together with 187 healthy controls were enrolled (all from the Chinese Han population in NE China). A fragment of 316 bp was amplified from the blood genome of each participant by polymerase chain reaction (PCR) using specific primers that targeted the Homo sapiens Wnt10a gene. By using the restriction enzyme AluI, the population analysed were classified into three genotypes, GG (316 bp), GA (316 bp, 117bp, 199bp) and AA (117bp, 199bp) based on the rs147680216 G/A polymorphism (Gly>Ser mutation at position 213 of Wnt10a protein) of theWnt10a gene. The frequency of allele A in the affected group was significantly higher (14.1% compared with 3.2% in the control group). The allele G with an odds ratio (OR) of 0.201 and 95% CI of 0.445 to 0.091 was not a risk factor for the condition in the affected group. However, the distribution of the genotype did affect its occurrence in the affected group (p<0.001), but not the classification of types (p=0.901). In conclusion we found an rs147680216 G>A mutation that was associated with non-syndromic cleft lip and palate in the Wnt10a gene.

Statistical analysis of disease-causing and neutral mutations in human membrane proteins.

Mutations in transmembrane proteins (TMPs) have diverse effects on their structure and functions, which may lead to various diseases. In this present study, we have investigated variations in human membrane proteins and found that negatively charged to positively charged/polar and nonpolar to nonpolar changes are dominant in disease-causing and neutral mutations, respectively. Further, we analyzed the top 10 preferred mutations in 14 different disease classes and found that each class has at least two Arg mutations. Moreover, in cardiovascular diseases and congenital disorders of metabolism, Cys mutations occur more frequently in single-pass proteins, whereas Arg and nonpolar residues are more frequently substituted in multi-pass membrane proteins. The immune system diseases are enriched in C → R and C → Y mutations in inside and outside regions. On the other hand, in the membrane region, E → K and R → Q mutations are prevalent. The comparison of mutations in topologically similar regions of globular and membrane proteins showed that Ser and Thr mutations cause deleterious effects in membrane regions, whereas Cys and charged residues, Asp and Arg are prevalent in the buried regions of globular proteins. Our comprehensive analysis of disease-associated mutations in transmembrane proteins will be useful for developing prediction tools.

The PTN-PTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway for oligodendrocyte differentiation: Targeted inactivation of PTPRZ activity in mice.

Protein tyrosine phosphatase receptor type Z (PTPRZ) maintains oligodendrocyte precursor cells (OPCs) in an undifferentiated state. The inhibition of PTPase by its ligand pleiotrophin (PTN) promotes OPC differentiation; however, the substrate molecules of PTPRZ involved in the differentiation have not yet been elucidated in detail. We herein demonstrated that the tyrosine phosphorylation of AFAP1L2, paxillin, ERBB4, GIT1, p190RhoGAP, and NYAP2 was enhanced in OPC-like OL1 cells by a treatment with PTN. AFAP1L2, an adaptor protein involved in the PI3K-AKT pathway, exhibited the strongest response to PTN. PTPRZ dephosphorylated AFAP1L2 at tyrosine residues in vitro and in HEK293T cells. In OL1 cells, the knockdown of AFAP1L2 or application of a PI3K inhibitor suppressed cell differentiation as well as the PTN-induced phosphorylation of AKT and mTOR. We generated a knock-in mouse harboring a catalytically inactive Cys to Ser (CS) mutation in the PTPase domain. The phosphorylation levels of AFAP1L2, AKT, and mTOR were higher, and the expression of oligodendrocyte markers, including myelin basic protein (MBP) and myelin regulatory factor (MYRF), was stronger in CS knock-in brains than in wild-type brains on postnatal day 10; however, these differences mostly disappeared in the adult stage. Adult CS knock-in mice exhibited earlier remyelination after cuprizone-induced demyelination through the accelerated differentiation of OPCs. These phenotypes in CS knock-in mice were similar to those in Ptprz-deficient mice. Therefore, we conclude that the PTN-PTPRZ signal stimulates OPC differentiation partly by enhancing the tyrosine phosphorylation of AFAP1L2 in order to activate the PI3K-AKT pathway.

Identification and Characterisation of a Novel Antioxidant Activity for the BCAT1 Cxxc Motif: Implications for Myeloid Leukaemia Development

Recently the human cytosolic branched-chain amino transferase (BCAT1) has been implicated in the development of myeloid leukaemia. Previously studies identified a redox active CXXC motif for BCAT1 analogous to the mitochondrial isoform (BCAT2). Oxidation of this CXXC motif can regulate aminotransferase activity in both isoforms. Interestingly the reduction mid-point potential (Em) of the BCAT1 CXXC motif is around 80mV lower compared with BCAT2. This suggests that the BCAT1 CXXC motif is more reducing in nature i.e. has antioxidant capacity. Further evidence to support this notion comes from the observation that BCAT1 is significantly less sensitive to inactivation mediated by hydrogen peroxide (H2O2). This is an important observation, since H2O2 is a reactive oxygen species (ROS) implicated in myeloid leukaemia development. Here we provide the first evidence to show that the BCAT1 CXXC can metabolise H2O2. By using CXXC motif mutant constructs, we also evaluate the role of the BCAT1 CXXC motif in myeloid leukaemia cells.To investigate the antioxidant capacity of the BCAT1 CXXC motif, cDNA (accession NM_001178094.1) was cloned into a pET28a vector for overexpression and purification in E.coli BL21(DE3) cells. Site directed mutagenesis of the CXXC motif subsequently followed creating three constructs: 1) BCAT1-WT (unmutated), 2) BCAT1-CXXS (C-terminal Cys → Ser mutant) and 3) BCAT1-SXXS (N/C-terminal Cys → Ser double mutant). Mutation of the BCAT1 CXXC motif was confirmed at the protein level by 5,5'-ditho-bis-(2-nitrobenzoic acid) titration. WT and CXXC motif mutant BCAT1 protein was added to 5mM H2O2 & monitored at λ240nm for the disappearance of H2O2. This was compared with 1U of catalase as a positive control. Our data show that 15μg of purified BCAT1-WT could metabolise H2O2 at a rate of 0.14±0.02 μmol/min. The BCAT1 CXXC motif mutants lacked capacity to do this. To verify our findings, BCAT1-WT treated with N-ethylmaleimide also lost the capacity to metabolise H2O2. This confirms that the antioxidant activity observed is Cys mediated. This novel finding for the BCAT1 CXXC motif may therefore be important in myeloid leukaemia development.To evaluate the BCAT1 CXXC motif in myeloid leukaemia cells, WT and CXXS mutant BCAT1 was subcloned into a pLENTI-C-Myc-DDK lentiviral vector prior to transduction of U937 cells. Empty vector (EV) control transgenic U937 cells were also generated. Stable expression of BCAT1 was achieved and confirmed by western-blot and qPCR; BCAT1-WT (6.5±1.7 fold increase) & BCAT1-CXXS (5.1±2.6 fold increase). The BCAT1 and EV transgenic U937 cells were subjected to H2O2 mediated oxidative stress and monitored for cell viability after 24h. A significant difference in the LD50 between BCAT1-WT, BCAT1-CXXS and EV control cells was observed; 620±28mM, 306±37mM, 251±26mM respectively (p<0.05). This finding suggests that the BCAT1 CXXC provides protection against H2O2 mediated cell death. To corroborate these findings, the same cells treated with rotenone observed a similar pattern in LD50; 1.05mM, 0.06mM, 0.12mM respectively.ROS are implicated in many cellular processes, including differentiation. Thus, we next asked whether the BCAT1 CXXC motif could suppress U937 cell differentiation mediated by phorbol 12-myristate 13-acetate (PMA). This compound was selected since ROS feature in PMA mediated differentiation. U937 cells were monitored for the expression of CD11b and CD36 following 48h PMA incubation. Our data show a significant reduction in the frequency of CD11b+/CD36+ U937 cells for BCAT1-WT (17.5±6.9%) compared with EV controls (54.0±10.5%, p<0.05). BCAT1-CXXS also showed a reduction (30.6±4.9%), but not to the same extent as BCAT1-WT. This finding was mirrored by the expression level of CD11b, where the MFI was significantly less for BCAT1-WT (88.0±16.3) compared with EV control (251.5±54.7, p<0.001) and BCAT1-CXXS (152.5±6.3, p<0.05). Analysis of intracellular ROS using 2′,7′-Dichlorodihydrofluorescein diacetate revealed a reduction in cellular ROS for BCAT1-WT cells (5929±13.8 MFI), compared with EV controls (8527±196 MFI) and BCAT1-CXXS (7879±18.4 MFI). This shows that the BCAT1 CXXC motif may control cellular ROS levels in myeloid leukaemia cells.In summary, this study identifies a novel antioxidant role for the BCAT1 CXXC which confers protection against ROS mediated cell death and differentiation of myeloid leukaemia cells. DisclosuresNo relevant conflicts of interest to declare.

Arabidopsis thaliana alcohol dehydrogenase is differently affected by several redox modifications.

In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.

Characterization of the Leaf Color Mutant hy and Identification of the Mutated Gene in Chinese Cabbage

Leaf color mutants play an important role in our understanding of chlorophyll biosynthesis and catabolism. In this study, we obtained a yellow-green leaf mutant hy in an ethyl methanesulfonate mutagenized population of chinese cabbage (Brassica rapa ssp. pekinensis). The hy phenotype was controlled by a recessive allele at a single locus. The intrinsic photochemical activity of photosystem II (PSII) is impaired in hy, suggesting that absorbed light energy is not efficiently transferred from the light-harvesting complexes antenna to the PSII reaction centers and dissipated as heat or fluorescence. We measured chlorophyll content and chlorophyll precursors and analyzed the expression of key genes in the chlorophyll synthetic pathway in hy and wild type. The mutation phenotype was consistent with inhibited expression of chlorophyll a oxygenase (CAO) gene in the chlorophyll synthetic pathway. In mutant hy, CAO cDNA was cloned so that a C to T mutation at 1099 bp caused a conserved proline (Pro) to serine (Ser) mutation at the 367th amino acid in C-domain, which changed the secondary structure of CAO protein. We speculate that the mutation amino acid changed in the C-domain may affect the catalytic function in mutant CAO.

Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

Tooth development is a complex process that involves precise and time-dependent orchestration of multiple genetic, molecular, and cellular interactions. Ameloblastin (AMBN, also named “amelin” or “sheathlin”) is the second most abundant enamel matrix protein known to have a key role in amelogenesis. Amelogenesis imperfecta (AI [MIM: 104500]) refers to a genetically and phenotypically heterogeneous group of conditions characterized by inherited developmental enamel defects. The hereditary dentin disorders comprise a variety of autosomal-dominant genetic symptoms characterized by abnormal dentin structure affecting either the primary or both the primary and secondary teeth. The vital role of Ambn in amelogenesis has been confirmed experimentally using mouse models. Only two cases have been reported of mutations of AMBN associated with non-syndromic human AI. However, no AMBN missense mutations have been reported to be associated with both human AI and dentin disorders. We recruited one kindred with autosomal-dominant amelogenesis imperfecta (ADAI) and dentinogenesis imperfecta/dysplasia characterized by generalized severe enamel and dentin defects. Whole exome sequencing of the proband identified a novel heterozygous C-T point mutation at nucleotide position 1069 of the AMBN gene, causing a Pro to Ser mutation at the conserved amino acid position 357 of the protein. Exfoliated third molar teeth from the affected family members were found to have enamel and dentin of lower mineral density than control teeth, with thinner and easily fractured enamel, short and thick roots, and pulp obliteration. This study demonstrates, for the first time, that an AMBN missense mutation causes non-syndromic human AI and dentin disorders.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Dissection of the Erv41‐Erv46 retrieval pathway suggests a redox‐regulated mechanism

The early secretory pathway consists of ER‐ and Golgi‐resident proteins involved in synthesis, folding, quality control, and post translational modifications of secretory proteins. Anterograde flux through this pathway can lead to the mislocalization of ER‐resident proteins as they passively leak to the Golgi. Fortunately there are several retrieval mechanisms to return mislocalized ER‐residents back to the ER. One such retrieval process identified by our lab relies on the Erv41‐Erv46 complex, which recognizes specific non‐HDEL ER‐residents in the Golgi and returns them to the ER in a COPI‐dependent manner. The focus of our work is to determine the mechanism by which the Erv41‐Erv46 retrograde receptor binds to escaped ER‐residents in the Golgi and releases this cargo in the ER.Site‐directed mutagenesis to introduce Erv41 and Erv46 mutations in Saccharomyces cerevisiae revealed key residues involved in retrieval of non‐HDEL ER‐residents. One such series of mutations involves a cysteine rich region of Erv46 where Cys to Ser mutants result in a complete loss of function. This cysteine rich region contains two CXXC motifs typically found in redox proteins. As observed in other redox proteins, the residues between the vicinal cysteines in this region of Erv46 are important suggesting that the redox potential of these cysteines is finely tuned for activity. Furthermore, mutations of a four residue hydrophobic patch within the cysteine rich region as well as a distal basic residue in Erv46 leads to a failure in retrieval of ER‐residents. These residues, but not the cysteines, are conserved in Erv41, however concomitant mutations in Erv41 do not affect retrieval suggesting Erv46 is predominantly involved in binding while Erv41 may play a distinct role.Based on our results, we propose the redox state of Erv46 is highly dynamic resulting in active and inactive disulfide configurations which can expose or mask a cargo binding hydrophobic patch. Defining the Erv41‐Erv46 binding mechanism may allow for a more complete understanding of ER homeostasis as well as providing a potential target to block pathogenic toxins that exploit Erv41‐Erv46 mediated retrieval for efficient delivery to the ER.Support or Funding InformationNIH GM52549This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancing cytochrome P450-mediated non-natural cyclopropanation by mutation of a conserved second-shell residue.

Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and -nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h . Here, we demonstrate that mutation of a ubiquitously conserved second-shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first-shell Cys to Ser mutations. Furthermore, we show that these mutations confer improved non-natural catalysis in 4 diverse P450 scaffolds.