scholarly journals Accounting for biases in riboprofiling data indicates a major role for proline and not positive amino acids in stalling translation

2014 ◽  
Author(s):  
Carlo G. Artieri ◽  
Hunter B. Fraser
Keyword(s):  
Amino Acids ◽  
Ribosome Profiling ◽  
Side Chain ◽  
Data Sets ◽  
Charged Amino Acids ◽  
Recent Method ◽  
Low Coverage ◽  
Positively Charged

The recent advent of ribosome profiling ? sequencing of short ribosome-bound fragments of mRNA ? has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling dataset have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a recent method that reported positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that its assumptions lead to false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling.

scholarly journals The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter

1999 ◽  
Vol 181 (5) ◽  
pp. 1524-1529 ◽  
Author(s):  
Paolo Landini ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Promoter Architecture ◽  
Charged Amino Acids ◽  
Identify Amino Acid ◽  
Ada Protein ◽  
Positively Charged

ABSTRACT The methylated form of the Ada protein (meAda) activates transcription from the Escherichia coli ada,aidB, and alkA promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit ς70 that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in ς70 region 4 results in decreased Ada-dependent binding of RNA polymerase to thealkA promoter in vitro and impairs alkAtranscription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-ς70interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307–13312, 1998), we showed that a set of negatively charged amino acids in ς70 region 4 is involved inmeAda-ς70 interaction at the adaand aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affectmeAda-dependent transcription at ada andaidB. Unlike the ς70 amino acids involved in the interaction with meAda at the ada andaidB promoters, K593, K597, and R603 are not conserved in ςS, an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. WhilemeAda is able to promote transcription by the ςS form of RNA polymerase (EςS) atada and aidB, it fails to do so atalkA. We propose that meAda can activate transcription at different promoters by contacting distinct determinants in ς70 region 4 in a manner dependent on the location of the Ada binding site.

scholarly journals Biosynthesis of willardiine and isowillardiine in germinating pea seeds and seedlings

1972 ◽  
Vol 129 (4) ◽  
pp. 897-905 ◽  
Author(s):  
T. S. Ashworth ◽  
E. G. Brown ◽  
F. M. Roberts
Keyword(s):  
Amino Acids ◽  
Side Chain ◽  
Free Base ◽  
Pyrimidine Metabolism ◽  
Uracil Derivatives ◽  
Weak Activity ◽  
Pea Seeds

The synthesis of the pyrimidinyl amino acids willardiine and isowillardiine was studied in vivo and in vitro. Uracil derivatives stimulate the biosynthesis of both compounds; the free base is the most effective. Significant incorporation of [2-14C]uracil and [6-14C]orotate into willardiine and isowillardiine was found. Incorporation of [6-14C]orotate was substantially decreased in the presence of uracil, and to a lesser extent by uridine and UMP. [3-14C]Serine was incorporated into the alanine side chain of the two uracilylalanines but not into the ring. The effect of a number of uracil analogues and inhibitors of pyrimidine metabolism was examined. Some were shown to stimulate the biosynthesis; the most noticeable effects were obtained with 6-azauracil and 2-thiouracil. Attempts to obtain extracts capable of synthesizing the uracilylalanines from uracil and serine were unsuccessful, but weak activity was observed when serine was replaced by O-acetylserine.

The VES KM: a pathway for protein folding in vivo

2020 ◽  
Vol 92 (1) ◽  
pp. 179-191
Author(s):  
Leonor Cruzeiro
Keyword(s):  
Amino Acids ◽  
Protein Folding ◽  
Protein Structures ◽  
Kinetic Mechanism ◽  
Thermal Bath ◽  
Charged Amino Acids ◽  
Nascent Chain ◽  
Dynamics Simulations ◽  
Positively Charged

AbstractWhile according to the thermodynamic hypothesis, protein folding reproducibility is ensured by the assumption that the native state corresponds to the minimum of the free energy in normal cellular conditions, here, the VES kinetic mechanism for folding in vivo is described according to which the nascent chain of all proteins is helical and the first and structure defining step in the folding pathway is the bending of that initial helix around a particular amino acid site. Molecular dynamics simulations are presented which indicate both the viability of this mechanism for folding and its limitations in the presence of a Markovian thermal bath. An analysis of a set of protein structures formed only of helices and loops suggests that bending sites are correlated with regions bounded, on the N-side, by positively charged amino acids like Lysine and Histidine and on the C-side by negatively charged amino acids like Aspartic acid.

PAK: an essential motif for forming β-turn structures and exhibiting the thrombolytic effect of P6A and its analogs

2007 ◽  
Vol 85 (6) ◽  
pp. 730-740 ◽  
Author(s):  
Ming Zhao ◽  
Xin Jia ◽  
Chao Wang ◽  
Qin Li ◽  
Kexiang Zhou ◽  
...  
Keyword(s):  
Side Chain ◽  
Conformational Structure ◽  
Thrombolytic Activity ◽  
Solution Structures ◽  
Structure Relationship ◽  
Thrombolytic Effect ◽  
Dependent Pathway ◽  
Positively Charged

Ala-Arg-Pro-Ala-Lys (ARPAK; also known as P6A) and 19 of its analogs were synthesized, and their thrombolytic activities were assessed in vitro and in vivo. The solution structures of 12 of the P6A analogs were determined using nuclear magnetic resonance (NMR) spectroscopy. The thrombolytic activity and conformational structure relationship was analyzed. We found that the Pro-Ala-Lys (PAK) sequence was essential for thrombolytic activity and was also responsible for the β-turn structure found in the P6A analogs studied. The well defined β turn may act as a binding head with the protruding lysine side-chain (positively charged) found at the target site for target recognition. Additionally, the N-terminal residue may be critical for thrombolytic activity, which for PAK-containing peptides, is likely achieved via a plasminogen-dependent pathway.

scholarly journals The role of amino acids in hydroxyapatite mineralization

2016 ◽  
Vol 13 (123) ◽  
pp. 20160462 ◽  
Author(s):  
M. Tavafoghi ◽  
M. Cerruti
Keyword(s):  
Amino Acids ◽  
Critical Role ◽  
Matrix Proteins ◽  
Mineral Density ◽  
Hormone Production ◽  
Charged Amino Acids ◽  
Collagen Mineralization ◽  
Positively Charged

Polar and charged amino acids (AAs) are heavily expressed in non-collagenous proteins (NCPs), and are involved in hydroxyapatite (HA) mineralization in bone. Here, we review what is known on the effect of single AAs on HA precipitation. Negatively charged AAs, such as aspartic acid, glutamic acid (Glu) and phosphoserine are largely expressed in NCPs and play a critical role in controlling HA nucleation and growth. Positively charged ones such as arginine (Arg) or lysine (Lys) are heavily involved in HA nucleation within extracellular matrix proteins such as collagen. Glu, Arg and Lys intake can also increase bone mineral density by stimulating growth hormone production. In vitro studies suggest that the role of AAs in controlling HA precipitation is affected by their mobility. While dissolved AAs are able to inhibit HA precipitation and growth by chelating Ca 2+ and PO 4 3− ions or binding to nuclei of calcium phosphate and preventing their further growth, AAs bound to surfaces can promote HA precipitation by attracting Ca 2+ and PO 4 3− ions and increasing the local supersaturation. Overall, the effect of AAs on HA precipitation is worth being investigated more, especially under conditions closer to the physiological ones, where the presence of other factors such as collagen, mineralization inhibitors, and cells heavily influences HA precipitation. A deeper understanding of the role of AAs in HA mineralization will increase our fundamental knowledge related to bone formation, and could lead to new therapies to improve bone regeneration in damaged tissues or cure pathological diseases caused by excessive mineralization in tissues such as cartilage, blood vessels and cardiac valves.

The 1- and 2-Naphthylalanine Analogs of Oxytocin and Vasopressin

1995 ◽  
Vol 60 (12) ◽  
pp. 2170-2177 ◽  
Author(s):  
Zdenko Procházka ◽  
Jiřina Slaninová
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Pressor Test

Solid phase technique on p-methylbenzhydrylamine resin was used for the synthesis of four analogs of oxytocin and four analogs of vasopressin with the non-coded amino acids L- or D- and 1- or 2-naphthylalanine and D-homoarginine. [L-1-Nal2]oxytocin, [D-1-Nal2]oxytocin, [L-2-Nal2]oxytocin, [D-2-Nal2]oxytocin, [L-1-Nal2, D-Har8]vasopressin, [D-1-Nal2, D-Har8]vasopressin, [L-2-Nal2, D-Har8]vasopressin and [D-2-Nal2, D-Har8]vasopressin were synthesized. All eight analogs were found to be uterotonic inhibitors in vitro and in vivo. Analogs with 2-naphthylalanine are stronger inhibitors, particularly in the vasopressin series than the analogs with 1-naphthylalanine. Analogs with 1-naphthylalanine have no activity in the pressor test, analogs with 2-naphthylalanine are weak pressor inhibitors.

scholarly journals Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1597
Author(s):  
Thuong Thi Ho ◽  
Van Thi Pham ◽  
Tra Thi Nguyen ◽  
Vy Thai Trinh ◽  
Tram Vi ◽  
...  
Keyword(s):  
High Pressure ◽  
Neutralizing Antibodies ◽  
H5n1 Virus ◽  
Vaccine Development ◽  
Particle Characterization ◽  
Innovative Strategy ◽  
Specific Igg ◽  
Positively Charged

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

scholarly journals Synthesis, Characterization and Evaluation of Peptide Nanostructures for Biomedical Applications

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4587
Author(s):  
Fanny d’Orlyé ◽  
Laura Trapiella-Alfonso ◽  
Camille Lescot ◽  
Marie Pinvidic ◽  
Bich-Thuy Doan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biomedical Applications ◽  
Chemical Reactivity ◽  
Physical Stability ◽  
Chemical Properties ◽  
Building Blocks ◽  
Imaging Probes ◽  
Therapeutic Molecules

There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.

Turnover of total proteins and ornithine aminotransferase during liver regeneration in rats

1980 ◽  
Vol 238 (1) ◽  
pp. E46-E52
Author(s):  
S. L. Augustine ◽  
R. W. Swick
Keyword(s):  
Amino Acids ◽  
Liver Regeneration ◽  
Protein Degradation ◽  
Partial Hepatectomy ◽  
Free Amino ◽  
Liver Protein ◽  
Ornithine Aminotransferase ◽  
Fractional Rate

The recovery of approximately 40% of the total liver protein during the first day after partial hepatectomy was shown to be due to the near cessation of protein breakdown rather than to an increase in protein synthesis. The decrease in degradation of total protein was less if rats were adrenalectomized or protein-depleted prior to partial hepatectomy. The effect of these treatments originally suggested that changes in free amino acid levels in liver might be related to the rate of protein degradation. However, no correlation was found between levels of total free amino acids and rates of breakdown. Measurements of individual amino acids during liver regeneration suggested that levels of free methionine and phenylalanine, amino acids that have been found to lower rates of protein degradation in vitro, are not correlated with rates of breakdown in vivo. The difference between the fractional rate of ornithine aminotransferase degradation (0.68/day and 0.28/day in sham-hepatectomized and partially hepatectomized rats, respectively) was sufficient to account for the higher level of this protein 3 days after surgery in the latter group.

scholarly journals The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

2009 ◽  
Vol 284 (24) ◽  
pp. 16317-16324 ◽  
Author(s):  
Sandra Mueller ◽  
Gunnar Kleinau ◽  
Mariusz W. Szkudlinski ◽  
Holger Jaeschke ◽  
Gerd Krause ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thyroid Stimulating Hormone ◽  
Hinge Region ◽  
Camp Production ◽  
Glycoprotein Hormone ◽  
Charged Amino Acids ◽  
Bovine Thyroid ◽  
Positively Charged ◽  
Bovine Tsh ◽  
Signaling Activity

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.

Sign in / Sign up

Export Citation Format

Share Document