Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein.

Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.

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Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54836-5 ◽

1991 ◽

Vol 266 (31) ◽

pp. 21174-21178

Author(s):

T. Watanabe ◽

N. Wada ◽

E.E. Kim ◽

H.W. Wyckoff ◽

J.Y. Chou

Keyword(s):

Alkaline Phosphatase ◽

Amino Acid ◽

Germ Cell ◽

Single Amino Acid ◽

Placental Alkaline Phosphatase

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Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

10.26434/chemrxiv.5972173.v1 ◽

2018 ◽

Author(s):

Nidhi Gour ◽

Bharti Koshti ◽

Chandra Kanth P. ◽

Dhruvi Shah ◽

Vivek Shinh Kshatriya ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Self Assembly ◽

Aromatic Amino Acids ◽

Neutral Condition ◽

Single Amino Acid ◽

Binding Assays ◽

Human Neuroblastoma ◽

Cytotoxicity Assays ◽

First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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Phylogenetic analysis and predicted functional effect of protein mutations of E6 and E7 HPV16 strains isolated in Indonesia

Medical Journal of Indonesia ◽

10.13181/mji.v24i4.1197 ◽

2015 ◽

Vol 24 (4) ◽

pp. 197-205

Author(s):

Dwi Wulandari ◽

Lisnawati Rachmadi ◽

Tjahjani M. Sudiro

Keyword(s):

Amino Acids ◽

Phylogenetic Analysis ◽

Amino Acid ◽

Asian American ◽

Protein Function ◽

Single Amino Acid ◽

Hpv16 E6 ◽

E6 And E7 ◽

Neutral Mutations

Background: E6 and E7 are oncoproteins of HPV16. Natural amino acid variation in HPV16 E6 can alter its carcinogenic potential. The aim of this study was to analyze phylogenetically E6 and E7 genes and proteins of HPV16 from Indonesia and predict the effects of single amino acid substitution on protein function. This analysis could be used to reduce time, effort, and research cost as initial screening in selection of protein or isolates to be tested in vitro or in vivo.Methods: In this study, E6 and E7 gene sequences were obtained from 12 samples of Indonesian isolates, which were compared with HPV16R (prototype) and 6 standard isolates in the category of European (E), Asian (As), Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and North American (NA) branch from Genbank. Bioedit v.7.0.0 was used to analyze the composition and substitution of single amino acids. Phylogenetic analysis of E6 and E7 genes and proteins was performed using Clustal X (1.81) and NJPLOT softwares. Effects of single amino acid substitutions on protein function of E6 and E7 were analysed by SNAP.Results: Java variants and isolate ui66* belonged to European branch, while the others belonged to Asian and African branches. Twelve changes of amino acids were found in E6 and one in E7 proteins. SNAP analysis showed two non neutral mutations, i.e. R10I and C63G in E6 proteins. R10I mutations were found in Af-2 genotype (AF472509) and Indonesian isolates (Af2*), while C63G mutation was found only in Af2*.Conclusion: E6 proteins of HPV16 variants were more variable than E7. SNAP analysis showed that only E6 protein of African-2 branch had functional differences compared to HPV16R.

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Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1247 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1247-1252 ◽

Cited By ~ 35

Author(s):

E Lazar ◽

S Watanabe ◽

S Dalton ◽

M B Sporn

Keyword(s):

Amino Acids ◽

Biological Activity ◽

Amino Acid ◽

Growth Factor ◽

Aspartic Acid ◽

Transforming Growth Factor ◽

Biologically Active ◽

Single Amino Acid ◽

Transforming Growth Factor Alpha ◽

Factor Alpha

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

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SOME ASPECTS OF THE STRUCTURE OF HEMOGLOBIN

Canadian Journal of Biochemistry ◽

10.1139/o64-087 ◽

1964 ◽

Vol 42 (6) ◽

pp. 755-762 ◽

Cited By ~ 26

Author(s):

David B. Smith

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Partial Amino Acid Sequence ◽

Heme Pocket ◽

Terminal Amino ◽

Polypeptide Chains ◽

Kinetics Of ◽

Β Chain

An outline of present ideas concerning the arrangement, folding, and chemistry of the polypeptide chains of hemoglobin is given with some references to present know ledge of myoglobin.New material includes a partial amino acid sequence of the β-chain of horse hemoglobin, details concerning the amino acids lining the heme pocket of horse hemoglobin, and the effects of carboxypeptidases A and B on horse oxy- and horse deoxy-hemoglobin. The kinetics of the latter reactions are not simple. The C-terminal amino acids are released more rapidly from the oxygenated form.

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Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3850-3859.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3850-3859

Author(s):

T A Coleman ◽

C Kunsch ◽

M Maher ◽

S M Ruben ◽

C A Rosen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Binding Specificity ◽

Single Amino Acid ◽

Nf Kappa B ◽

Dna Binding Specificity ◽

Dna Motif ◽

Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3470 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3470-3480 ◽

Cited By ~ 98

Author(s):

E Moran ◽

B Zerler ◽

T M Harrison ◽

M B Mathews

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Amino Acid Position ◽

Cysteine Residue ◽

Transformation Function ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.281.4.g1034 ◽

2001 ◽

Vol 281 (4) ◽

pp. G1034-G1043 ◽

Cited By ~ 31

Author(s):

Kousei Ito ◽

Hiroshi Suzuki ◽

Yuichi Sugiyama

Keyword(s):

Amino Acids ◽

Multidrug Resistance ◽

Amino Acid ◽

Membrane Vesicles ◽

Single Amino Acid ◽

Sf9 Cells ◽

Transport Activity ◽

Wild Type ◽

Cationic Amino Acids ◽

The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

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Unusual Aggregation Properties of Single Amino Acid L-Lysine Hydrochloride

10.26434/chemrxiv.14152976 ◽

2021 ◽

Author(s):

Bharti Koshti ◽

Ramesh Singh ◽

Vivekshinh Kshtriya ◽

Shanka Walia ◽

Dhiraj Bhatia ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Self Assembly ◽

Short Term Memory ◽

Deionized Water ◽

The Body ◽

The Self ◽

Single Amino Acid ◽

Maple Syrup ◽

Self Assembling

. The self-assembly of single amino acids is very important topic of research since there are plethora of diseases like phenylketonuria, tyrosinemia, hypertryptophanemia, hyperglycinemia, cystinuria and maple syrup urine disease to name a few which are caused by the accumulation or excess of amino acids. These are in-born errors of metabolisms (IEM’s) which are caused due to the deficiency of enzymes involved in catabolic pathways of these enzymes. Hence, it is very pertinent to understand the fate of these excess amino acids in the body and their self-assembling behaviour at molecular level. From the previous literature reports it may be surmised that the single amino acids like Phenylalanine, Tyrosine, Tryptophan, Cysteine and Methionine assemble to amyloid like structures, and hence have important implications in the pathophysiology of IEM’s like phenylketonuria, tyrosinemia, hypertryptophanemia, cystinuria and hypermethioninemia respectively. In this manuscript we report the self-assembly of lysine hydrocholride to fiber like structures in deionized water. It could be observed that lysine assemble to globular structures in fresh condition and then gradually changes to fiber like morphologies by self-association over time after 24 hours. These fibers gradually change to tubular morphologies after 3 day followed by fractal irregular morphologies in 10 and 15 days respectively. Notably, lysine exists as positively charged amino acid at physiological pH and the amine groups in lysine remain protonated. Hence, the self-assembling properties of lysine hydrochloride in deionized water is also pertinent and give insights into the fate of this amino acid in body in case it remains unmetabolized. Further, MTT assays were done to analyse the toxicities of these aggregates and the assay suggest their cytotoxic nature on SHSY5Y neural cell lines. Hence, the aggregation of lysine may be attributed to the pathological symptoms caused in diseases like hyperlysinemia which is associated with the neurological problems like seizures and short-term memory as observed in case of amyloid diseases like Parkinson’s and Alzheimer’s to name a few.

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