Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors.     

Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

Zymocin is a Kluyveromyces lactis protein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells'' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α''s chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ''s capability to deliver other cargo proteins into target cells.     

Site-Directed Mutagenesis Evidence for Arginine-384 Residue at the Active Site of Maize Branching Enzyme II

Essential arginine residues are suggested to be located at the active sites of maize branching enzymes (BE) based on the evidence that two arginine residues are conserved in all BE from various species and that as little as one arginine residue is located at the active site of maize BE by phenylglyoxal (PGO) modification. To determine the exact location of the active-site arginine residue in BE, we employed peptide mapping and site-directed mutagenesis approaches. A single trypsin-digested, [¹⁴C]PGO-labeled peptide was purified from maize BEII by two rounds of HPLC separation, but we failed to obtain amino acid sequencing information. Site-directed mutagenesis was then used to create one mutant (arginine-384 to alanine-384), R384A. Immunoblotting result showed that BEII protein was expressed at a similar level in the wild type and the R384A mutant. However, BE activity in the R384A mutant was only 1.4% of the wild type. These results support the conclusion that the conserved arginine-384 residue is important in BEII catalysis.     

Molecular Modeling and Site-Directed Mutagenesis Reveal Essential Residues for Catalysis in a Prokaryote-Type Aspartate Aminotransferase

We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the “open-to-closed” transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.     

~(21)Ala定点突变胰高血糖素及结构与功能变化

 胰高血糖素是由 2 9个氨基酸组成的多肽激素 ,具有促糖元分解的生理功能 ,其拮抗剂有治疗糖尿病病人的潜在应用价值 .在获得重组胰高血糖素基因工程菌基础上 ,利用定点突变技术改造其第 2 1位氨基酸天冬氨酸为丙氨酸 ,并经DNA测序证明胰高血糖素基因发生了点突变 .用IPTG诱导表达后 ,经亲和层析和反相高效液相层析 ,纯化到突变型重组2 1Ala 胰高血糖素 .质谱测定分子量与理论值相符 .利用园二色谱比较重组胰高血糖素和突变的2 1Ala 胰高血糖素在TFE中的二级结构 ,发现胰高血糖素以α螺旋为主要二级结构 ,2 1Ala 胰高血糖素仍有α螺旋结构特征 ,并且含量有所增大 .利用兔升血糖试验 ,发现2 1Ala 胰高血糖素生物活性比重组胰高血糖素减少 51 % (P <0 .0 1 ) .显示天然胰高血糖素第 2 1位氨基酸天冬氨酸与形成α螺旋结构关系不大 ,但在发挥胰高血糖素的生物功能中有重要作用 ,与其可作为钙离子结合位点 ,参与胰高血糖素和受体结合的潜在功能密切相关 .     

Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168–203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.     

Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.     

Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein

Molecular determinants of virulence in flaviviruses cluster in two regions on the three-dimensional structure of the envelope (E) protein; the base of domain II, believed to serve as a hinge during pH-dependent conformational change in the endosome, and the lateral face of domain III, which contains an integrin-binding motif Arg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to form the receptor-binding site of the protein. In an effort to better understand the nature of attenuation caused by mutations in these two regions, a full-length infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was employed to produce a panel of site-directed mutants with substitutions at amino acid positions 277 (E-277; hinge region) or 390 (E-390; RGD motif). Viruses with mutations at E-277 (Ser-->Ile, Ser-->Asn, Ser-->Val, and Ser-->Pro) showed various levels of in vitro and in vivo attenuation dependent on the level of hydrophobicity of the substituted amino acid. Altered hemagglutination activity observed for these viruses suggests that mutations in the hinge region may indirectly disrupt the receptor-ligand interaction, possibly by causing premature release of the virion from the endosomal membrane prior to fusion. Similarly, viruses with mutations at E-390 (Asp-->Asn, Asp-->Glu, and Asp-->Tyr) were also attenuated in vitro and in vivo; however, the absorption and penetration rates of these viruses were similar to those of wild-type virus. This, coupled with the fact that E-390 mutant viruses were only moderately inhibited by soluble heparin, suggests that RGD-dependent integrin binding is not essential for entry of MVE and that multiple and/or alternate receptors may be involved in cell entry.     

The Role of the Strictly Conserved Positively Charged Residue Differs among the Gram-positive,Gram-negative,and Chloroplast YidC Homologs

Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3 to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. Our results also show that the C-terminal half of the helical hairpin structure in cytoplasmic loop C1 is important for the activity of YidC because various deletions in the region either eliminate or impair YidC function. The results here underscore the importance of the cytoplasmic hairpin region for YidC and show that the arginine is critical for the tested Gram-positive YidC homolog but is not essential for the tested Gram-negative and chloroplast YidC homologs.     

Crucial Positively Charged Residues for Ligand Activation of the GPR35 Receptor

GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the β-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the β-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.     

Stabilization of Penicillin G Acylase from Escherichia coli: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4°C, pH 5 at 60°C, pH 7 at 55°C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.     

Random Transposon-Mediated Mutagenesis of the Essential Large Tegument Protein pUL36 of Pseudorabies Virus

Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.The herpesvirus particle is composed of four structural elements. The DNA genome-containing core is enclosed in an icosahedral capsid, which, in turn, is embedded in a proteinaceous layer termed the tegument and enveloped by a cell-derived membrane containing viral glycoproteins (35). The tegument of the Alphaherpesvirinae contains more than 15 different viral and several cellular proteins and can be structurally and functionally separated into at least two layers: a capsid-proximal “inner” part and an envelope-associated “outer” part (reviewed in references 34 and 35). The largest tegument proteins in all herpesviruses analyzed so far are homologs of herpes simplex virus type 1 (HSV-1) pUL36, which are essential for viral replication. pUL36, its interaction partner, pUL37, and the pUS3 kinase are part of the inner tegument and remain associated with nucleocapsids during their transport along microtubules to the nuclear pore (2, 3, 19, 31). In contrast, other tegument proteins like pUL46, pUL47, and pUL49 rapidly diffuse in the cytoplasm after fusion of the virion envelope with the plasma membrane. Proteolytic cleavage of HSV-1 pUL36 after docking of the nucleocapsid to the nuclear pore appears to be required for release of viral DNA into the nucleus (22). Besides these roles early in infection, pUL36 also functions during later stages of replication in virion maturation. After assembly in the nucleus, nucleocapsids are translocated to the cytoplasm by budding at the inner nuclear membrane and fusion with the outer nuclear membrane (34). Although functional nuclear localization motifs have been described for pseudorabies virus (PrV) and HSV-1 pUL36 (1, 37), in PrV-infected cells, pUL36 was never detected in the nucleus but was added to nascent virions early after nuclear egress (18, 27, 31, 37). It has been suggested that pUL36 interacts either directly (9, 32, 42, 44) or indirectly via capsid-associated pUL25 (10) with the capsid shell starting the tegumentation process in the cytosol.In PrV, pUL36 is the only tegument protein which has been shown to be truly essential. It consists of 3,084 amino acids (aa), resulting in a molecular mass of more than 300 kDa (27). Deletion of pUL36 in HSV-1 and PrV abolished viral replication. Ultrastructurally, similar phenotypes with nonenveloped nucleocapsids present in the cytoplasm and the lack of extracellular particles indicated a defect in virion maturation in the cytoplasm (13, 16). Several functional domains have been identified in pUL36. The interaction domain of pUL36 with pUL37 (5, 16, 20, 27, 36, 42) could be located in the N-terminal part of PrV and HSV-1 pUL36 (16, 36) (Fig. ​(Fig.1).1). Deletion of the pUL37 binding site in PrV pUL36 (PrV-UL36BSF) resulted in a similar phenotype to deletion of pUL37 with an impairment of secondary envelopment in the cytoplasm (16, 26). Unlike in PrV, pUL37 is essential for replication in HSV-1 (14, 30).Open in a separate windowFIG. 1.Schematic overview of PrV pUL36 and corresponding insertion mutants. (A) Diagram of the PrV genome with the unique long (U_L) and unique short (U_S) regions as well as repeat regions (internal repeat, IR; terminal repeat, TR). The positions of BamHI restriction sites are indicated, and restriction fragments are numbered according to their size. (B) Schematic diagram of the UL36 open reading frame with conserved regions. Pfam analysis (4; http://www.sanger.ac.uk/Software/Pfam/) delineated two highly conserved PfamA domains within pUL36 homologs of herpesviruses of all three herpesvirus subfamilies [box I, Herpes_teg_N PrV (p)UL36, aa 11 to 178] and of alphaherpesviruses [box II, Herpes_UL36 PrV (p)UL36, aa 1000 to 1251] as well as PfamB domains (hatched rectangles) (6) (C) Known essential and nonessential regions in PrV pUL36. Nonessential regions are shown in gray, with the positions of the amino acids deleted in the corresponding constructs (6, 8). Deletions tested by Lee et al. (28) are shown below, marked by arrows. The essential C terminus is shown in black. Besides the N-terminal deletion Δ6-225, none of the truncated proteins was functional. (D) Predicted or identified motifs in pUL36: USP (Cys26), active-site cysteine of the deubiquitinating activity (24); pUL37 interaction domain (16, 27); NLS, nuclear localization signal (37); leucine zipper (27); and late domain motifs PPKY and PSAP (6). (E) Locations of linker insertions in pUL36 are indicated by arrows and the position of the amino acid immediately preceding the insertion. Insertions shown by arrows pointing upwards yielded functional proteins, while arrows pointing downwards indicate nonfunctional mutants. Insertions resulting in proteins which were impaired but not fully deficient in complementation are underlined. For orientation, the BamHI site separating BamHI fragments 1 and 2 is indicated.A second functional domain in the N terminus of pUL36 comprises a ubiquitin-specific cysteine protease (USP) activity which could be identified in all three herpesvirus subfamilies (24, 40, 41). Interestingly, the USP activity is not essential for virus replication in cell culture (7, 21, 25, 43). However, it is relevant for oncogenicity of Marek′s disease virus (MDV) (21) and for virion maturation and neuroinvasion of PrV (7, 8, 29).Several other regions in PrV pUL36 were deleted without abolishing virus replication (6, 8, 28). While deletion of nearly 1/3 of the protein in the C-terminal part (aa 2087 to 2981) had only a slight effect, deletion of a region containing two leucine zipper motifs impaired virus replication and spread more strongly (8). The highly conserved C-terminal 62 amino acids, except for the extreme C-terminal 6 amino acids, are essential for virus replication (6, 28). Due to the size of the protein, a more detailed mutagenesis analysis has, however, not yet been undertaken.Therefore, the aim of our study was to construct random insertion mutants of PrV pUL36 using transposon-mediated insertion mutagenesis resulting in a 5-amino-acid linker insertion. Mutant proteins were analyzed functionally in transient transfection assays for complementation, and stable recombinants were isolated and further characterized.     

线粒体抗病毒蛋白对先天免疫的影响

病毒感染启动宿主先天免疫反应是通过激活转录因子NF-κB和干扰素调节因子3（IRF-3）,它们协同调控Ⅰ型干扰素的表达。病毒在复制过程中产生的复制中间体双链RNA作为一个病原相关分子模式,被细胞内具有RNA解旋酶活性的维甲酸诱导基因Ⅰ（RIG-1）编码蛋白检测到。线粒体抗病毒蛋白（MAVS）作为一个接头蛋白,在RIG-1信号通路的下游和NF-κB、IRF-3信号通路的上游扮演着重要的角色。MAVS通过其疏水跨膜结构域定位在线粒体外膜上,是线粒体中发现的第一个与先天免疫相关的蛋白质,将线粒体和先天免疫联系在一起。