Asn Mutation Research Articles

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of 6 amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together these data allow us to develop a general model for activation of TpoR and to explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Analisis Kebijakan Mutasi Asn Pasca Pilkada Berdasarkan Undang-Undang Nomor 10 Tahun 2016 Perspektif Fiqh Siyasah

Mutations for the State Civil Apparatus are commonplace in an organization or government agency. However, the mutation policy that was carried out after the Pilkada in Muna Regency was considered a technical, administrative and political phenomenon. The aims of this study are (1) to identify the mechanism for post-election mutations in Muna Regency; (2) Identifying the legal implications of the ASN mutation policy after the Regional Head Election in the Muna Regency government based on Article 72 paragraph (2) of Law number 10 of 2016 (3) Identifying Fiqh Siyasah's views regarding the ASN Mutation Policy after the Regional Head Election in Regional Government Muna District. The results of this study indicate that (1) In practice, the mutations carried out after the Regional Head Election are based on the needs of employees in a State Civil Apparatus employee office, this is done for the distribution of State Civil Apparatus Employees evenly and fairly; (2) Implementation of mutations according to the law, mutations are carried out on functional officials while what is meant in Article 72 of Law number 10 of 2016 are structural officials and functional officials who get additional tasks; (3 Mutation policy in Fiqh Siyasah can be done and occurred during the time of the Prophet Muhammad until the Caliphate of Umar Bin Al-Khattab.

Analisis Pelaksanaan Mutasi Aparatur Sipil Negara Eselon III Dan IV Lingkup Pemerintah Kabupaten Buton Utara

This study aims to analyze the implementation of echelon III and IV ASN mutations in the North Buton Regency Government. This study uses a qualitative approach, with an analysis focus on the implementation of echelon III and IV ASN mutations. There were 9 informants in this research, namely the Regional Secretary, Head of BKPSDM, Head of Mutation Division, 3 echelon III officials and 3 echelon IV officials. The selected informants were determined purposively. Data were collected through observation, interviews, and document studies. The data obtained were then analyzed interactively with an analytical model referring to Milles and Huberman (1992), covering data collection, data presentation, data reduction and drawing conclusions. The results showed that the implementation of the transfer of echelon III and IV employees within the scope of the North Buton Regency Government was still dominated by the spoil system. The mutations carried out are based on the principle of kinship and on the basis of likes and dislikes. These likes and dislikes are closely related to the behavior of employees who make political contributions to the implementation of democratic momentum in North Buton Regency. However, mutations based on considerations of the merid system and spoiled system are still the main considerations in the implementation of echelon III and IV ASN mutations in North Buton Regency.

Effect of Competency and Job Placement on Performance with Job Satisfaction as Intervening Variable (Study at The Regional Civil Service Agency Of Bondowoso Regency)

This research is motivated by several gaps or differences in research results with expert opinions, also supported by some research problem data, namely based on ASN mutation data showing that there are still many ASN whose work placements are not in accordance with their field competencies. Based on the results of the identification of employee performance analysis in 2020, it shows that the realization of ASN work in Bondowoso Regency has not been maximized. The purpose of this study was to determine directly or indirectly the effect of employee competence and job placement on employee performance through job satisfaction as an intervening variable for all structural employees of the Bondowoso Regency Government. The population of this study was 3,051 employees, while the number of samples was 354 using purposive sampling technique. The analysis technique used is the Structural Equation Model (SEM) using WarpPLS 5.0. The results of data analysis show that employee competence and job placement have a significant effect on job satisfaction, (2) employee competence and job placement and job satisfaction have a significant effect on employee performance, (2) employee competence and work placement have a significant effect on employee performance through job satisfaction as intervention variables.

Effect of Competency and Job Placement on Performance with Job Satisfaction as Intervening Variable (Study at the Regional Civil Service Agency of Bondowoso Regency)

<p><em>This research is motivated by several gaps or differences in research results with expert opinions, also supported by some research problem data, namely based on ASN mutation data showing that there are still many ASN whose work placements are not in accordance with their field competencies. Based on the results of the identification of employee performance analysis in 2020, it shows that the realization of ASN work in Bondowoso Regency has not been maximized. The purpose of this study was to determine directly or indirectly the effect of employee competence and job placement on employee performance through job satisfaction as an intervening variable for all structural employees of the Bondowoso Regency Government. The population of this study was 3,051 employees, while the number of samples was 354 using purposive sampling technique. The analysis technique used is the Structural Equation Model (SEM) using WarpPLS 5.0. The results of data analysis show that employee competence and job placement have a significant effect on job satisfaction, (2) employee competence and job placement and job satisfaction have a significant effect on employee performance, (2) employee competence and work placement have a significant effect on employee performance through job satisfaction as intervention variables.</em></p>

The consensus Nglyco -X-S/T motif and a previously unknown Nglyco -N-linked glycosylation are necessary for growth and pathogenicity of Phytophthora.

Asparagine (Asn, N)-linked glycosylation within Nglyco -X-S/T; X≠P motif is a ubiquitously distributed post-translational modification that participates in diverse cellular processes. In this work, N-glycosylation inhibitor was shown to prevent Phytophthora sojae growth, suggesting that N-glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map N-glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cyst, and sexual oospore developmental stages. A total of 355 N-glycosylated proteins was found, containing 496 glycosites, potentially involved in glycan degradation, carbon metabolism, glycolysis, or other metabolic pathways. Through PNGase F deglycosylation assays and site-directed mutagenesis of a GPI transamidase protein (GPI16) upregulated in cysts and a heat shock protein 70 (HSP70) upregulated in oospores, we demonstrated that both proteins were N-glycosylated and that the Nglyco -N motif is a target site for asparagine - oligosaccharide linkage. Glycosite mutations of Asn 94 Nglyco -X-S/T in the GPI16 led to impaired cyst germination and pathogenicity, while mutation of the previously unknown Asn 270 Nglyco -N motif in HSP70 led to decreased oospore production. In addition to providing a map of the oomycete N-glycoproteome, this work confirms that P. sojae has evolved multiple N-glycosylation motifs essential for growth.

Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine.

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

Case report: Analysis of BRCA1 and BRCA2 gene mutations in a hereditary ovarian cancer family

ObjectiveBreast cancer susceptibility gene 1/2 (BRCA1/2) is the most important susceptibility gene associated with hereditary ovarian cancer (HOC). We aimed to screen BRAC1 and BRAC2 gene mutations in a member of a hereditary ovarian cancer family in China, and to analyze the structure and function of the mutant protein.MethodsA typical HOC family was selected. Blood samples and pathological tissue samples were taken from the female members of the family. Blood samples from two patients with sporadic ovaries of the same pathological type were taken as a control group. After RNA extraction, PCR amplification was applied and the PCR products were directly sequenced and aligned, prediction and analysis of protein structure and molecular conformation that may be caused by BRCA1/2 mutation.ResultsThe whole gene analysis of BRCA1 and BRCA2 in ovarian cancer patients in the family showed that there were 8 mutations in BRCA1 whole gene sequencing, including 3 nonsense mutations (2314C>T, 2543T>C, 4540T>C); two mutations have been recorded, which are associated with cervical cancer (2844C>T) and endometriosis (3345A>G); three newly discovered mutations (3780A>G, 5069A>G, 3326A>T). Among them, 3780A>G and 5069A>G caused amino acid changes, while 3326A>T mutation caused Arg mutation to stop codon. A total of 7 mutations were detected in BRCA2 whole-genome sequencing, including 5 non-significant mutations (3623A>G, 4034T>C, 4790A>G, 6740G>C, 7469A>G); one no-record mutation (1716T>A), and 1 recorded mutation (1342A>C), which was associated with breast cancer and ovarian cancer. BRCA1 (3326A>T) and BRCA2 (1342A>C) mutations were co-existing in patients (II1, II3, and II5) identified as serous adenocarcinoma grade II. Two cases of ovarian serous cystadenocarcinoma with no history of family tumors were normalized for BRCA1/2 gene sequencing. In the gene detection of III generation female, four females with BRCA2 (1342A>C) mutation were found, and one of them also carried the BRCA1 (3326A>T) mutation, who can be considered a high-risk group of HOC in this family. Online protein structure predictions revealed that BRCA1 (3326A>T) mutations mutated AGA at this site to TGA resulting in a translated Arg (arginine) mutation as a stop codon, while BRCA2 (1342A>C) mutated AAT at this site to CAT resulting in a translated Asn mutation to His.ConclusionThe BRCA1 (3326A>T) and BRCA2 (1342A>C) were detected in the HOC family, which may be the susceptibility gene of the family’s HOC. The BRCA1/2 gene screening may be possible to obtain high-risk populations in this family.

Impact of co-existence of PMQR genes and QRDR mutations on fluoroquinolones resistance in Enterobacteriaceae strains isolated from community and hospital acquired UTIs

BackgroundFluoroquinolones are commonly recommended as treatment for urinary tract infections (UTIs). The development of resistance to these agents, particularly in gram-negative microorganisms complicates treatment of infections caused by these organisms. This study aimed to investigate antimicrobial resistance of different Enterobacteriaceae species isolated from hospital- acquired and community-acquired UTIs against fluoroquinolones and correlate its levels with the existing genetic mechanisms of resistance.MethodsA total of 440 Enterobacteriaceae isolates recovered from UTIs were tested for antimicrobial susceptibility. Plasmid-mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were examined in quinolone-resistant strains.ResultsAbout (32.5%) of isolates were resistant to quinolones and (20.5%) were resistant to fluoroquinolones. All isolates with high and intermediate resistance phenotypes harbored one or more PMQR genes. QnrB was the most frequent gene (62.9%) of resistant isolates. Co-carriage of 2 PMQR genes was detected in isolates (46.9%) with high resistance to ciprofloxacin (CIP) (MICs > 128 μg/mL), while co-carriage of 3 PMQR genes was detected in (6.3%) of resistant isolates (MICs > 512 μg/mL). Carriage of one gene only was detected in intermediate resistance isolates (MICs of CIP = 1.5–2 μg/mL). Neither qnrA nor qnrC genes were detected. The mutation at code 83 of gyrA was the most frequent followed by Ser80-Ile in parC gene, while Asp-87 Asn mutation of gyrA gene was the least, where it was detected only in high resistant E. coli isolates (MIC ≥128 μg/mL). A double mutation in gyrA (Lys154Arg and Ser171Ala) was observed in high FQs resistant isolates (MIC of CIP < 128 μg/mL).ConclusionFQs resistance is caused by interact between PMQR genes and mutations in both gyrA and parC genes while a mutation in one gene only can explain quinolone resistance. Accumulation of PMQR genes and QRDR mutations confers high resistance to FQs.

Sequencing of bulks of segregants allows dissection of genetic control of amylose content in rice.

SummaryAmylose content (AC) is a key quality trait in rice. A cross between Oryza glaberrima (African rice) and Oryza sativa (Asian rice) segregating for AC was analysed by sequencing bulks of individuals with high and low AC. SNP associated with the granule bound starch synthase (GBSS1) locus on chromosome 6 were polymorphic between the bulks. In particular, a G/A SNP that would result in an Asp to Asn mutation was identified. This amino acid substitution may be responsible for differences in GBSS activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. Other polymorphisms in genomic regions closely surrounding this variation may be the result of linkage drag. In addition to the variant in the starch biosynthesis gene, SNP on chromosomes 1 and 11 linked to AC was also identified. SNP was found in the genes encoding the NAC and CCAAT‐HAP5 transcription factors that have previously been linked to starch biosynthesis. This study has demonstrated that the approach of sequencing bulks was able to identify genes on different chromosomes associated with this complex trait.

Molecular Insights into the Interaction Between the SPRY Domain-Containing SOCS Box Protein SPSB2 and Peptides Based on the Binding Motif from iNOS

SPRY domain-containing SOCS box proteins SPSB1, 2, and 4 mediate the proteasomal degradation of inducible nitric oxide synthase (iNOS) and thereby modulate the amount of NO available for combating infectious organisms. A highly conserved Asp-Ile-Asn-Asn-Asn (DINNN) motif found at the N-terminus of iNOS binds to SPSB2 with nanomolar affinity. The design of specific and potent inhibitors of iNOS–SPSB interactions will be aided by a better understanding of the interactions of this DINNN sequence with SPSB2. Although crystal structures of SPSB complexes with DINNN peptides are available, aspects of the interaction between peptide and protein are still not fully understood. Here, our results from surface plasmon resonance and NMR spectroscopy indicate that residues flanking the DINNN motif, which make no direct contact with SPSB2 in the available crystal structures, nonetheless play an important role in enhancing the binding affinity to SPSB2, by up to 80-fold. Mutational analysis of the DINNN sequence showed that mutation of the Asp or the first Asn residue to Ala reduced the binding affinity by 200- or 600-fold respectively, whereas mutation of the third Asn made binding undetectable. Ala substitution of the second Asn residue caused a 30-fold drop in binding affinity. Substitution of the Ile had very little effect on the binding affinity and substitutions with bulky residues were tolerated. This provides an opportunity for further modification for therapeutic applications. These results highlight the complex interplay of peptide sequence and protein binding and inform efforts to design peptide therapeutics to disrupt the iNOS–SPSB interaction.

The combination of sequence-specific and nonspecific DNA-binding modes of transcription factor SATB1.

Transcription factor SATB1 (special AT-rich sequence binding protein 1) contains multiple DNA-binding domains (DBDs), i.e. two CUT-domain repeats (CUTr1 and CUTr2 from the N-terminus) and a homeodomain, and binds to the matrix attachment region (MAR) of DNA. Although CUTr1 and the homeodomain, but not CUTr2, are known to contribute to DNA binding, different research groups have not reached a consensus on which DBD is responsible for recognition of the target sequence in MAR, 5'-TAATA-3'. Here, we used isothermal titration calorimetry to demonstrate that CUTr1 has binding specificity to this motif, whereas the homeodomain shows affinity for a variety of DNAs without specificity. In line with nonspecific DNA-binding properties of the homeodomain, a mutation of the invariant Asn at position 51 of the homeodomain (typically in contact with the A base in a sequence-specific binding mode) did not affect the binding affinity significantly. The NMR analyses and computational modeling of the homeodomain, however, revealed the tertiary structure and DNA-binding mode that are typical of homeodomains capable of sequence-specific binding. We believe that the lack of highly conserved basic residues in the helix relevant to the base recognition loosens its fitting into the DNA groove and impairs the specific binding. The two DBDs, when fused in tandem, showed strong binding to DNA containing the 5'-TAATA-3' motif with an affinity constant >10(8) M(-1) and retained nonspecific binding activity. The combination of the sequence-specific and nonspecific DNA-binding modes of SATB1 should be advantageous in a search for target loci during transcriptional regulation.

Improving the Biodesulfurization of Crude Oil and Derivatives: A QM/MM Investigation of the Catalytic Mechanism of NADH-FMN Oxidoreductase (DszD).

The development of biocatalytic desulfurization strategies of petroleum and its derivatives could result in more economic alternatives than the widely used chemical desulfurization. The organism Rhodococcus erythropolis IGTS8 has been shown to metabolize organic sulfur compounds through a mechanism known as 4S pathway, which involves four enzymes (DszA, DszB, DszC, and DszD) and has been explored in biodesulfurization. Here we have applied QM/MM methods to study the catalytic mechanism of the enzyme DszD, a NADH-FMN oxidoreductase that occupies a central place on the 4S pathway by catalyzing the formation of the FMNH2 that is used by the two monooxynases in the cycle: DszA and DszC. In addition, to clarify the catalytic mechanism of this enzyme, this study analyzed in detail the role played by the active site Thr residue and of Asn and Ala enzyme mutants. The results help to explain previous experimental evidence and suggest new strategies for improving biodesulfurization through an increase in the activity of DszD.

His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His(499) near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His(499) is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His(499) regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca(2+) channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca(2+) channels.

Changing a conserved amino acid in R2R3-MYB transcription repressors results in cytoplasmic accumulation and abolishes their repressive activity in Arabidopsis.

Sub-group 4 R2R3-type MYB transcription factors, including MYB3, MYB4, MYB7 and MYB32, act as repressors in phenylpropanoid metabolism. These proteins contain the conserved MYB domain and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) repression domain. Additionally, MYB4, MYB7 and MYB32 possess a putative zinc-finger domain and a conserved GY/FDFLGL motif in their C-termini. The protein 'sensitive to ABA and drought 2' (SAD2) recognizes the nuclear pore complex, which then transports the SAD2-MYB4 complex into the nucleus. Here, we show that the conserved GY/FDFLGL motif contributes to the interaction between MYB factors and SAD2. The Asp → Asn mutation in the GY/FDFLGL motif abolishes the interaction between MYB transcription factors and SAD2, and therefore they cannot be transported into the nucleus and cannot repress their target genes. We found that MYB4(D261N) loses the capacity to repress expression of the cinnamate 4-hydroxylase (C4H) gene and biosynthesis of sinapoyl malate. Our results indicate conservation among MYB transcription factors in terms of their interaction with SAD2. Therefore, the Asp → Asn mutation may be used to engineer transcription factors.

Structure-function analysis and genetic interactions of the Yhc1, SmD3, SmB, and Snp1 subunits of yeast U1 snRNP and genetic interactions of SmD3 with U2 snRNP subunit Lea1.

Yhc1 and U1-C are essential subunits of the yeast and human U1 snRNP, respectively, that stabilize the duplex formed by U1 snRNA at the pre-mRNA 5′ splice site (5′SS). Mutational analysis of Yhc1, guided by the human U1 snRNP crystal structure, highlighted the importance of Val20 and Ser19 at the RNA interface. Though benign on its own, V20A was lethal in the absence of branchpoint-binding complex subunit Mud2 and caused a severe growth defect in the absence of U1 subunit Nam8. S19A caused a severe defect with mud2▵. Essential DEAD-box ATPase Prp28 was bypassed by mutations of Yhc1 Val20 and Ser19, consistent with destabilization of U1•5′SS interaction. We extended the genetic analysis to SmD3, which interacts with U1-C/Yhc1 in U1 snRNP, and to SmB, its neighbor in the Sm ring. Whereas mutations of the interface of SmD3, SmB, and U1-C/Yhc1 with U1-70K/Snp1, or deletion of the interacting Snp1 N-terminal peptide, had no growth effect, they elicited synthetic defects in the absence of U1 subunit Mud1. Mutagenesis of the RNA-binding triad of SmD3 (Ser-Asn-Arg) and SmB (His-Asn-Arg) provided insights to built-in redundancies of the Sm ring, whereby no individual side-chain was essential, but simultaneous mutations of Asn or Arg residues in SmD3 and SmB were lethal. Asn-to-Ala mutations SmB and SmD3 caused synthetic defects in the absence of Mud1 or Mud2. All three RNA site mutations of SmD3 were lethal in cells lacking the U2 snRNP subunit Lea1. Benign C-terminal truncations of SmD3 were dead in the absence of Mud2 or Lea1 and barely viable in the absence of Nam8 or Mud1. In contrast, SMD3-E35A uniquely suppressed the temperature-sensitivity of lea1▵.

The Mu Subunit of Plasmodium falciparum Clathrin-Associated Adaptor Protein 2 Modulates In Vitro Parasite Response to Artemisinin and Quinine

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of phenylalanine. It thus appears that the second-coordination sphere Asp residue in cPAH, and, by extrapolation, the equivalent residue in other AAAHs, plays a role in fine-tuning pterin affinity in the ground state via deformable interactions with bridging waters and assumes a more significant role in the transition state by aligning the cofactor through direct hydrogen bonding.