Changes In Amino Acid Sequence Research Articles

Method to Identify Silent Codon Mutations That May Alter Peptide Elongation Kinetics and Co-translational Protein Folding.

Due to the redundancy of the protein genetic code, mutational changes in the second or third nucleotide of an existing codon may not change the amino acid specification of the resulting modified codon. When peptide primary sequence is unchanged by mutation, that mutation is assumed to have no functional consequences. However, for one key gene involved in drug transport, MDR-1, several silent, synonymous mutations have been shown to alter protein structure and substrate affinity (Kimchi-Sarfaty et al., Science 315:525-528, 2007). The mechanism of these changes, in the absence of primary amino acid sequence changes, appears to be the change in abundance of the transfer RNA molecules complementary to the mutated, although synonymous, new codon. Transfer RNA abundance is proportional to the frequency of each codon as found in human protein coding DNA (Sharp et al., Nucleic Acids Res 14(13):5125-5143, 1986). These frequencies have been mapped for many thousands of human proteins (Nakamura et al., Nucleic Acids Res 28:292, 2000). This method analyzes silent codon mutations in whole genome data. Where there are large changes in codon frequency resulting from codon sequence mutation, the affected proteins are mapped to potential disease pathways, in the context of clinical phenotypes associated with the patient genome data.

A Novel Somatic Mutation in IKZF1 Is Common in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Myelofibrosis

Background:Myeloproliferative neoplasms (MPN) including polycythemia vera, essential thrombocytosis, and primary and secondary myelofibrosis (MF) are characterized by driver mutations in JAK2, CALR, and MPL. Additional somatic variants can be seen. Currently allogeneic hematopoietic stem cell transplant (HCT) is the only treatment with curative potential, but it is associated with a high risk of mortality, exceeding 50% in some patient subsets. The risk of disease progression usually outweighs the risk of transplant in patients with advanced MF. Here, we have evaluated the mutational profile using a targeted next-generation sequencing panel of 54 genes involved in myeloid neoplasms in patients with MF treated with HCT.Patients and Methods:48 patients with a diagnosis of primary (n=18) or secondary (n=30) MF who received a HCT at our center and from whom genomic DNA was available from pre-HCT marrow aspirates were evaluated. DNA was isolated from whole marrow aspirates obtained prior to HCT. Paired-end indexed libraries were sequenced at an average read depth of 1000x on Illumina MiSeq platform. Sequences were aligned to hg19 using the Burrows-Wheeler Aligner, and nucleotide variants were identified using the Genome Analysis Toolkit and Picard tools. Variants were annotated with ANNOVAR. Variants present at >5% variant allele frequency (VAF) with at a minimum of 25 variant reads were selected for further analysis. Variants were called "mutations" if they resulted in (1) non-synonymous changes in amino acid sequence, including nonsense and missense mutations, (2) the variant was known to be associated with hematologic malignancies in COSMIC database, or (3) the variant was present at < 0.5% in 1000 genomes and ExAC databases. Comparisons between clinical variables and mutations were analyzed with chi-square tests, adjusted for multiple testing.Results:The average age of the 48 patients was 55 years (range 23-72) and 52% of patients were female. Disease risk was estimated with DIPSS-Plus (High 33%, INT-2 53%, INT-1 7%, Low 7%). Unfavorable cytogenetics were present in 27% of patients. Donors included a matched sibling donor in 30% (n=14), a matched unrelated donor in 60% (n=29), and an alternative donor in 10% (n=5). At 3 years, overall survival was 52%, 17% of patients had relapsed disease, and 31% had non-relapse mortality.In our cohort, 38% had a mutation in CALR (n=15 type-I and n=3 type-II), 29% in JAK2 (n=14), 0% in MPL, and 33% were triple-negative (n=16; no evidence of CALR, JAK2, or MPL mutation). The average VAF of CALR mutations was 50% (range 20-82%) and JAK2 mutations was 58% (range 25-81%). In addition to driver mutations, 94% of patients had at least one other mutation in 26 of the 54 genes sequenced. On average, patients had 2.1 other mutations (range 0-5 mutations in addition to driver mutation). The most common other mutations were in ASXL1 (31%), IKZF1 (23%), ETV6 (17%), and U2AF1 (17%). The VAF of these other mutations spanned 9-76%.Patients with triple-negative MF were more likely to have primary MF (p=0.019) and platelets <100 (p<0.001), than patients with JAK2 and CALR mutations. Patients with ≥3 other mutations were more likely to have hemoglobin <10 (p=0.02) and a higher rate of relapse (p<0.01) than patients with <3 other mutations. Patients with ASXL1 mutations were more likely to have peripheral blood blasts (p<0.001).Mutations in ASXL1, ETV6, and U2AF1 have been previously reported in myeloid malignancies. Like other groups, we found ASXL1 mutations associated with worse prognosis. Here we report a novel, recurrent mutation in the gene IKZF1. All patients with IKZF1 mutation carried the same insertion of 11 nucleotides leading to an early frameshift and ultimate nonsense mutation (L92fs), which would lead to deletion of the zinc-finger domain. Such a mutation likely leads to loss of IKZF1 tumor suppressor function.Conclusions:Somatic mutations, in addition to driver mutations, are common in patients with MF treated with HCT. Mutation profiling in this patient subset may improve relapse risk prediction. Loss of heterozygosity on chromosome 7 mapping to the IKZF1 locus has been found by others to be associated with leukemic transformation in patients with MPN (Jaeger et al. 2010. Leukemia. 24: 1290). Given their frequency in this high risk MF population, further study of the role of IKZF1 mutations in MPN is warranted. DisclosuresRadich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; TwinStrand: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy.

Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth.

A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.

Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein

Retinal degeneration 3 (RD3) protein, essential for normal expression of retinal membrane guanylyl cyclase (RetGC) in photoreceptor cells, blocks RetGC catalytic activity and stimulation by guanylyl cyclase-activating proteins (GCAPs). In a mouse retina, RD3 inhibited both RetGC1 and RetGC2 isozymes. Photoreceptors in the rd3/rd3 mouse retinas lacking functional RD3 degenerated more severely than in the retinas lacking both RetGC isozymes, consistent with a hypothesis that the inhibitory activity of RD3 has a functional role in photoreceptors. To map the potential target-binding site(s) on RD3, short evolutionary conserved regions of its primary structure were scrambled and the mutations were tested for the RD3 ability to inhibit RetGC1 and co-localize with the cyclase in co-transfected cells. Substitutions in 4 out of 22 tested regions, (87)KIHP(90), (93)CGPAI(97), (99)RFRQ(102), and (119)RSVL(122), reduced the RD3 apparent affinity for the cyclase 180-700-fold. Changes of amino acid sequences outside the Lys(87)-Leu(122) central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the (93)CGPAI(97) portion of a predicted central α-helix most drastically suppressed the inhibitory activity of RD3 and disrupted RD3 co-localization with RetGC1 in HEK293 cells. Different side chains replacing Cys(93) profoundly reduced RD3 affinity for the cyclase, irrespective of their relative helix propensities. We conclude that the main RetGC-binding interface on RD3 required for the negative regulation of the cyclase localizes to the Lys(87)-Leu(122) region.

Genomic analysis of missense and silent mutations in CatSper 2 gene in infertile male cases.

Background: Infertility is described as inability of a couple to conceive after a period of one year regular unprotected intercourse. About 10% - 15% of couples experience some form of infertility. Male infertility is a multifactorial disease process with a number of potential contributing causes; several factors may contribute to its etiology. Cation channels of sperm is a small family of ion channels, normally referred to as CatSper channels (CatSper1, 2, 3 & 4) which are putative six-transmembrane (6TM) spanning proteins and seem to be specific to sperm cells. CatSper 2 gene involved in sperm motility and hyper activation. Materials and methods: We performed mutational analysis of the CatSper2 gene in 100 infertile males and 100 healthy controls. To screen mutations inCatSper2 gene, we performed PCR followed bydirect DNA sequencing. Results: Analysis of mutational screening of CatSper2 gene, we found a single nucleotide change (CT G →CT A ) in five asthenozoospermia at exon 3 resulting in silent mutation and (C G T→C C T)in eight asthenozoospermia at exon 6 missense mutation as the change leads to amino acid sequence change from Arginine to Proline. Conclusions: On mutational screening of CatSper2 gene, at exon 6 there was missense mutation that leads to conformational structural change inspite of the amino acids being hydrophobic and non-polar in nature. The identified mutations may be a cause of asthenozoospermia in humans.

Biomedical Mutation Analysis (BMA): A software tool for analyzing mutations associated with antiviral resistance

Introduction: Hepatitis C virus (HCV) is considered a major public health problem, with 200 million people infected worldwide. The treatment for HCV chronic infection with pegylated interferon alpha plus ribavirin inhibitors is unspecific; consequently, the treatment is effective in only 50% of patients infected. This has prompted the development of direct-acting antivirals (DAA) that target virus proteins. These DAA have demonstrated a potent effect in vitro and in vivo; however, virus mutations associated with the development of resistance have been described. Objective: To design and develop an online information system for detecting mutations in amino acids known to be implicated in resistance to DAA. Materials and methods: We have used computer applications, technological tools, standard languages, infrastructure systems and algorithms, to analyze positions associated with resistance to DAA for the NS3, NS5A, and NS5B genes of HCV. Results: We have designed and developed an online information system named Biomedical Mutation Analysis (BMA), which allows users to calculate changes in nucleotide and amino acid sequences for each selected sequence from conventional Sanger and cloning sequencing using a graphical interface. Conclusion: BMA quickly, easily and effectively analyzes mutations, including complete documentation and examples. Furthermore, the development of different visualization techniques allows proper interpretation and understanding of the results. The data obtained using BMA will be useful for the assessment and surveillance of HCV resistance to new antivirals, and for the treatment regimens by selecting those DAA to which the virus is not resistant, avoiding unnecessary treatment failures. The software is available at: http://bma.itiud.org.

Analysis of the cytochrome c oxidase subunit 1 (COX1) gene reveals the unique evolution of the giant panda

As the rate-limiting enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX) plays a crucial role in biological metabolism. “Living fossil” giant panda (Ailuropoda melanoleuca) is well-known for its special bamboo diet. In an effort to explore functional variation of COX1 in the energy metabolism behind giant panda's low-energy bamboo diet, we looked at genetic variation of COX1 gene in giant panda, and tested for its selection effect. In 1545 base pairs of the gene from 15 samples, 9 positions were variable and 1 mutation leaded to an amino acid sequence change. COX1 gene produces six haplotypes, nucleotide (pi), haplotype diversity (Hd). In addition, the average number of nucleotide differences (k) is 0.001629±0.001036, 0.8083±0.0694 and 2.517, respectively. Also, dN/dS ratio is significantly below 1. These results indicated that giant panda had a low population genetic diversity, and an obvious purifying selection of the COX1 gene which reduces synthesis of ATP determines giant panda's low-energy bamboo diet. Phylogenetic trees based on the COX1 gene were constructed to demonstrate that giant panda is the sister group of other Ursidae.

NV gene variations of viral hemorrhagic septicemia virus decrease cellular energy metabolism

The viral hemorrhagic septicemia virus (VHSV) is a viral pathogen to cause severe losses in farming industry of olive flounder. Among six-encoding VHSV proteins, NV protein has been shown to have an impact on virulence. Thirteen VHSV strains are isolated from olive flounders showing VHS symptoms and mortality in Korea, and then molecular variation of nucleotide and amino acid sequences of the NV genes were compared. Although common mutations were detected at four sites in nucleotide sequences of NV gene, there was no difference in the amino acid sequences among 13 VHSV variants. We identified substitution of one base of 34 positions within the NV ORF of 13 VHSV isolates and these point mutations resulted in changes in amino acid sequences at 6 residues of NV (8, 56, 81, 88, 117, and 119). Based on finding of NV-mediated decrease of intracellular ATP level, protein expression of six NV variants in flounder cells decreased more than control NV protein. The strongest decreasing activity on cellular ATP level was shown by No. 56 (serine→leucine) of NV variation. The variation changes the hydropathy index significantly. The results imply that we can use appearance of some amino acid variations as virulence markers in VHSV-derived mortality. Statement of relevance1. Recently VHSV has infected >80 kinds of fish species. The number of infection host of VHSV is rapidly increased. Among the VHSV-infected fishes, olive flounder is one of the most important fish of aquaculture in Korea, Japan, and China. In every year, we found the massive VHSV-derived mortality of olive flounder aquaculture in Korea. To overcome this VHSV pathogenesis, we need to identify high-pathogenic VHSV appearance at aquaculture in earlier time.2. Since VHSV gene is highly mutated in time-dependent manner, the efficient vaccine for VHSV-derived disease prevention is not well achieved. Therefore, the other approaches for prevention of epidemics by VHSV should be required. Our research goal is to develop prevention of epidemics propagation of VHSV infection by high pathogenic VHSV prediction with gene variation analysis.3. In this study, we identified several gene variations of VHSV NV protein from 13 VHSV specimens of severe VHS-showing olive flounders in Korea. The gene variations are associated with amino acid changes of VHSV NV proteins. The amino acid variants of NV protein show the inhibitory activity of fish cellular energy (ATP) generation. The cellular energy deprivation might give a big impact on fish health and mortality. The DNA mutation on specific amino acid changes is possibly applied to develop the PCR diagnosis system for early detection of high pathogenic VHSV appearance. The early pathogenic gene diagnosis of VHSV gives more efficient prevention of epidemics.

S1 gene sequence analysis of infectious bronchitis virus vaccinal strains (H120 & H52) and their embryo-passaged derivatives

Avian infectious bronchitis is an acute and highly contagious disease that mainly causes respiratory symptoms in poultry. A number of serotypes and variants of the viral agent with poor cross-protection are the major problem to achieve desired immunity from vaccination. The S1 subunit of S glycoprotein (spike) is the major determinant of IBV so that a minor change in amino acid sequence of this protein, alters the virus strain. Therefore, characterization of the sequence of S1 gene is necessary to identify virus strains and their similarities with the vaccinal strains. In this research, the S1 sequence of H52 and H120 vaccinal strains of Razi Institute was fully characterized, and also the effect of serial passages in embryonated - eggs (5 passages beyond the master seed) on the S1 gene was investigated. The results showed that H120 and H52 strains of Razi Institute are 100% identical to the reference vaccine strains available in the GenBank. In addition, the H52 strain showed one amino acid substitution from the 3rd passage in which Glycine (G) was replaced by Valine (V) at position 118 making these passages exactly identical to the H120 strain while no change occurred for the H120 strain during these passages. Analysis of the original vaccinal strains which are widely administered in Iran, is definitely useful for prevention and control strategies against the circulating viruses. To identify the genetic change(s) responsible for attenuation of these strains during passages in embryonated-egg, characterization of other genes, especially those involved in replication is recommended.

Asymmetric Functional Conversion of Eubacterial Light-driven Ion Pumps

In addition to the well-known light-driven outward proton pumps, novel ion-pumping rhodopsins functioning as outward Na(+) and inward Cl(-) pumps have been recently found in eubacteria. They convert light energy into transmembrane electrochemical potential difference, similar to the prototypical archaeal H(+) pump bacteriorhodopsin (BR) and Cl(-) pump halorhodopsin (HR). The H(+), Na(+), and Cl(-) pumps possess the conserved respective DTE, NDQ, and NTQ motifs in the helix C, which likely serve as their functional determinants. To verify this hypothesis, we attempted functional interconversion between selected pumps from each category by mutagenesis. Introduction of the proton-pumping motif resulted in successful Na(+) → H(+) functional conversion. Introduction of the respective characteristic motifs with several additional mutations leads to successful Na(+) → Cl(-) and Cl(-) → H(+) functional conversions, whereas remaining conversions (H(+) → Na(+), H(+) → Cl(-), Cl(-) → Na(+)) were unsuccessful when mutagenesis of 4-6 residues was used. Phylogenetic analysis suggests that a H(+) pump is the common ancestor of all of these rhodopsins, from which Cl(-) pumps emerged followed by Na(+) pumps. We propose that successful functional conversions of these ion pumps are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Dependence of the observed functional conversions on the direction of evolution strongly suggests that the essential structural mechanism of an ancestral function is retained even after the gain of a new function during natural evolution, which can be evoked by a few mutations. By contrast, the gain of a new function needs accumulation of multiple mutations, which may not be easily reproduced by limited mutagenesis in vitro.

Structural Role of Hydrophobic Core in Proteins-Selected Examples

This paper discusses the sequence/structure relation. The core question concerns the degree to which similar sequences produce similar structures and vice versa. A mechanism by which similar sequences may result in dissimilar structures is proposed, based on the Fuzzy Oil Drop (FOD) model in which structural similarity is estimated by analyzing the protein’s hydrophobic core. We show that local changes in amino acid sequences, in addition to producing local structural alterations at the substitution site, may also change the shape of the hydrophobic core, significantly affecting the overall tertiary conformation of the protein. Our analysis focuses on four sets of proteins: 1) Pair of designer proteins with specially prepared sequences; 2) Pair of natural proteins modified (mutated) to converge to a point of high-level sequence identity while retaining their respective wild-type tertiary folds; 3) Pair of natural proteins with common ancestry but with differing structures and biological profiles shaped by divergent evolution; and 4) Pair of natural proteins of high structural similarity with no sequence similarity and different biological function.

Molecular Basis for Distinct, Antagonistic, Regulatory Functions of Ribosomal Protein Paralogs Rpl22 and Rpl22-Like1 (Like1) in Hematopoiesis

The prevailing view of ribosomal proteins (RP) is that they act to facilitate the core function of the ribosome in synthesizing proteins. However, emerging evidence suggests that some RP perform regulatory functions outside of the context of the ribosome ("extra-ribosomally"). Indeed, our laboratory demonstrated that ribosomal protein, Rpl22 and its paralog Rpl22l1 (Like1), are dispensable for ribosome assembly and translation, yet they perform critical regulatory functions in hematopoiesis that are mediated by binding selected RNA targets and controlling their translation. Despite their high degree of homology, Rpl22 and Like1 antagonistically control the emergence of hematopoietic stem cells (HSC) by regulating the translation of the key transcription factor, Smad1, with Rpl22 repressing its translation and Like1 acting to oppose that translation. Here, we seek to establish the molecular basis for how such highly homologous proteins perform distinct and opposing functions. Since both Rpl22 and Like1 share highly conserved RNA-binding domains in the central core of the proteins, we hypothesize that their unique regulatory functions are likely to reside in their termini. To systematically test this hypothesis, we employed a domain-swapping approach, creating a series of Rpl22:Like1 chimeric proteins to assess which changes would confer on Like1, the Rpl22 function of blocking HSC emergence. In doing so, we utilized zebrafish as a model organism. During embryonic zebrafish development, the enforced expression of Rpl22, but not Like1, represses Smad1 expression and blocks HSC) emergence. Interestingly, our analysis of the Rpl22:Like1 chimeras revealed that transfer of the N-terminal amino acids of Rpl22 to Like1 was sufficient to confer upon Like1 the Rpl22 function of blocking HSC emergence. Moreover, this Rpl22:Like1 N-terminal chimera also was able to restore the block in T lymphocyte development observed upon loss of Rpl22, indicating that Rpl22 function in another hematopoietic process, supporting T lymphocyte development, can be conferred upon Like1 by the N-terminal sequences of Rpl22. Together, these data suggest that the unique and opposing functions of Rpl22 and Like1 reside in their N-termini. How these newly identified N-terminal amino acid sequences confer distinct biological functions on these two paralogs is unclear at present, but may involve the association with distinct co-factors. This possibility is currently under investigation. This study demonstrates that relative minor changes in amino acid sequence can have profound effects on the functions of these RP paralogs, enabling them to play divergent regulatory roles in controlling hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Investigation of biochemical changes of the ovine calpain 3 exon-10 polymorphism

Calpain 3 (CAPN3) is a tissue specific calpain, and its mRNA is the most expressed calpain isoform in skeletal muscles. Many mutations and polymorphisms within the human CAPN3 gene have been reported and related to limb-girdle muscular dystrophy. Several reports link CAPN3 polymorphisms and meat quality. An association between three allele variants in exon-10 of ovine CAPN3 and the yield of fat trimmed meat cuts has been reported. This research investigated the biochemical significance of polymorphic variation in CAPN3. CAPN3 mRNA sequences were obtained from muscle samples collected from lambs which were homozygous for each of the three alleles. Four single base substitutions were found besides those in exon-10, but none of them, including the variations within exon-10, caused a change in amino acid sequence. The expression of CAPN3 mRNA and the amounts of CAPN3 protein were also compared among genotypes, and no significant differences were found. These results suggest that the reported association of specific allele variants within CAPN3 exon-10 to phenotype variations were not direct effects of CAPN3 polymorphisms. Interspecies analyses of the CAPN3 sequences indicated that the sequence reported here is more likely to be the correct common ovine CAPN3 sequence than the reference sequence.

Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike “classical” primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of “classical” ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9–11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2–3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity.

Sintesis Tiga Peptida Bergugus Pelindung sebagai Prekursor Komponen Vaksin Influenza Universal

Current highly effective conventional vaccine to halt the spread of bird flu has not been invented yet because of susceptiblemutation of influenza virus. In spite of undergoing mutation which causes the amino acid sequence change, influenzaviruses maintain conservation at ectodomain of M2 protein, especially M2e(2-16) (SLLTEVETPIRNEW). The use ofconserved epitope M2e(2-16) in epitope-based vaccine potentially produces universal influenza vaccine. In designingepitope-based vaccine, the M2e(2-16) needs to be coupled with T helper epitope, P25, which is subsequently mentioned asM2e(2-16)-K-P25 (SLLTEVETPIRNEWGKKKL IPNASLIENCTKAEL). The M2e(2-16)-K-P25 was synthesized usingconvergent solid phase peptide synthesis strategy because of the size of the sequence. In this strategy, four peptideprecursors of M2e(2-16)-K-P25; SLLTEVETP (F1), IRNEWGK (F2), KLIPNASLI (F3), and ENCTKAEL (F4); were synthesizedin advance. After the precursors ready, coupling reaction was performed to obtain M2e(2-16)-K-P25. In the previousresearch, F3 has been obtained in high purity through Fmoc/tBu solid phase peptide synthesis method. In this conductedresearch, the three remaining precursors; F1, F2, and F4; were synthesized by the same method. Each peptide was analysedby thin layer chromatography, HPLC, and mass spectroscopy methods. F1, F2 and F4 were successfully synthesized andeach of them was detected at 1490.0, 1874.8 and 1881.9 amu, respectively. However, F1 was not possible to purify becauseof its insolubility in various solvents.

Antimicrobial Activity of Fosfomycin-Tobramycin Combination against Pseudomonas aeruginosa Isolates Assessed by Time-Kill Assays and Mutant Prevention Concentrations.

The antibacterial activity of fosfomycin-tobramycin combination was studied by time-kill assay in eight Pseudomonas aeruginosa clinical isolates belonging to the fosfomycin wild-type population (MIC = 64 μg/ml) but with different tobramycin susceptibilities (MIC range, 1 to 64 μg/ml). The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined in five of these strains (tobramycin MIC range, 1 to 64 μg/ml) in aerobic and anaerobic conditions simulating environments that are present in biofilm-mediated infections. Fosfomycin-tobramycin was synergistic and bactericidal for the isolates with mutations in the mexZ repressor gene, with a tobramycin MIC of 4 μg/ml. This effect was not observed in strains displaying tobramycin MICs of 1 to 2 μg/ml due to the strong bactericidal effect of tobramycin alone. Fosfomycin presented higher MPC values (range, 2,048 to >2,048 μg/ml) in aerobic and anaerobic conditions than did tobramycin (range, 16 to 256 μg/ml). Interestingly, the association rendered narrow or even null MSWs in the two conditions. However, for isolates with high-level tobramycin resistance that harbored aminoglycoside nucleotidyltransferases, time-kill assays showed no synergy, with wide MSWs in the two environments. glpT gene mutations responsible for fosfomycin resistance in P. aeruginosa were determined in fosfomycin-susceptible wild-type strains and mutant derivatives recovered from MPC studies. All mutant derivatives had changes in the GlpT amino acid sequence, which resulted in a truncated permease responsible for fosfomycin resistance. These results suggest that fosfomycin-tobramycin can be an alternative for infections due to P. aeruginosa since it has demonstrated synergistic and bactericidal activity in susceptible isolates and those with low-level tobramycin resistance. It also prevents the emergence of resistant mutants in either aerobic or anaerobic environments.

Histone variants: the artists of eukaryotic chromatin.

The eukaryotic genome is packaged into a complex nucleoprotein structure named chromatin, balancing the compactness of genome and the accessibility of regulatory proteins and RNA polymerases to DNA. The mechanisms of the regulation of chromatin dynamics include the post-translational modification of histones, alteration of nucleosome positions by chromatin remodelers, replacement of canonical histones by histone variants with the aid of histone chaperones, and dynamic organization of the three-dimensional genome in the small nucleus. Histone variants are different from canonical histones by substitution of several amino acid residues or changes in amino acid sequence. Histone variants perform specialized functions such as altering nucleosome stability, dynamics, structure, as well as playing critical roles in a range of biological processes like trans-criptional regulation, DNA repair and recombination, development and immune responses. Here we discuss how histone variants, their modification and specific loading to chromatin are involved in transcriptional regulation, DNA repair and plant development.

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

Rational design of hyper-glycosylated interferon beta analogs: A computational strategy for glycoengineering

Glycoengineering has been successfully used to improve the physicochemical and pharmaceutical properties of therapeutics. One aspect of glycoengineering is to introduce new N-linked glycosylation consensus sequences (Asn, X, Thr/Ser) into desirable positions in the peptide backbone by mutational insertion to generate proteins with increased sialic acid content. In the current work, human interferon beta (huIFN-β) was used as a model to identify the potential positions for the addition of new N-glycosylation sites. A computational strategy was employed to predict the structural distortions and functional alterations that might be caused by the change in amino acid sequence. Accordingly, three-dimensional (3D) structures of the designed huIFN-β analogs were generated by comparative modeling. Molecular dynamics (MD) simulation was carried out to assess the molecular stability and flexibility profile of the structures. Subsequently, for the purpose of glycoengineering huIFN-β, analogs with 3D structures more similar to the wild-type huIFN-β and exposed Asn residue in the new N-glycosylation site were identified. These modeling procedures indicated that the addition of the new N-glycosylation site in the loop regions had lower constraining effects on the tertiary structure of the protein. This computational strategy can be applied to avoid alterations in the 3D structure of proteins caused by changes in the amino acid sequences, when designing novel hyper-glycosylated therapeutics. This in turn reduces labor-intensive experimental analyses of each analog.