RNAi-mediated silencing of vitellogenin gene curtails oogenesis in the almond moth Cadra cautella

Vitellogenins, major yolk protein precursors, play an essential role in the reproduction and spread of all oviparous species, including insects. To investigate reproductive strategies of the warehouse moth Cadra cautella at the molecular level, a partial transcript of the C. cautella vitellogenin (CcVg) gene was extended through the rapid amplification of cDNA ends PCR and sequenced. The complete CcVg mRNA transcript was 5,334 bp long, which encoded a protein of 1,778 amino acids, including the first 14 amino acids of the signal peptide. The deduced CcVg protein contained a putative cleavage site (RTRR) at the amino-terminal side, similar to several other insect species. DGQR and GI/LCG motifs were present at the CcVg gene C-terminus, followed by nine cysteine residues. CcVg harbored 131 putative phosphorylation sites, numbering 84, 19, and 28 sites for serine, threonine, and tyrosine, respectively. The transcript showed a great resemblance with other lepidopteran Vgs. CcVg protein analysis revealed three conserved regions: 1) vitellogenin-N domain, 2) DUF 1943 (domain of unknown function), and 3) a von Willebrand factor type D domain. Additionally, sex, stage-specific, and developmental expression profiles of the CcVg gene were determined through RT-PCR. The Vg was first expressed in 22-day-old female larvae, and its expression increased with growth. The phylogenetic analysis based on different insect Vgs revealed that the CcVg exhibited close ancestry with lepidopterans. The CcVg-based RNAi experiments were performed, and the effects were critically evaluated. The qRT-PCR results showed that CcVg-based dsRNA suppressed the Vg gene expression up to 90% at 48 h post-injection. Moreover, CcVg-based RNAi effects resulted in low fecundity and egg hatchability in the CcVg-based dsRNA-treated females. The females laid eggs, but because of insufficient yolk protein availability the eggs could not succeed to hatch. The significant difference in the fecundity and hatchability unveils the importance of CcVg gene silencing and confirmed that the Vg gene plays a key role in C. cautella reproduction and it has the potential to be used as a target for RNAi-mediated control of this warehouse pest.

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Homochirality May Not Be a Prerequisite for Protein Structure and Function: A Computational Model Investigation of Prebiotic Peptides

10.20944/preprints202003.0005.v1 ◽

2020 ◽

Author(s):

Jianxun Shen ◽

Pauline M. Schwartz ◽

Carl Barratt

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Hierarchical Cluster ◽

Building Blocks ◽

Beta Sheets ◽

Folding Energy ◽

Primitive Earth ◽

C Terminus ◽

Significant Difference ◽

Prebiotic Amino Acids

On the primitive Earth, both L- and D-amino acids would have been present. However, only L-amino acids are essential blocks to construct proteins in modern life. To study the relative stability of homochiral and heterochiral peptides, a variety of computational methods were employed. 10 prebiotic amino acids (Gly, Ala, Asp, Glu, Ile, Leu, Pro, Ser, Thr, and Val) were previously determined by multiple previous meteorite, spark discharge, and hydrothermal vent studies. We focused on what had been reported as primary early Earth polypeptide analogs: 1ARK, 1PPT, 1ZFI, and 2LZE. Tripeptide composed of only Asp, Ser, and Val exemplified that different positions (i.e., N-terminus, C-terminus, and middle) made a difference in minimal folding energy of peptides, while the classification of amino acid (hydrophobic, acidic, or hydroxylic) did not show significant difference. Hierarchical cluster analysis for dipeptides with all possible combinations of the proposed 10 prebiotic amino acids and their D-amino acid substituted derivatives generated five clusters. Prebiotic polypeptides were built up to test the significance of molecular fluctuations, secondary structure occupancies, and folding energy differences based on these clusters. Most interestingly, among 129 residues, mutation sensitivity profiles presented that the ratio of more stable to less stable to equally stable D-amino acids was about 1:1:1. In conclusion, some combinations of a mixture of L- and D-amino acids can act as essential building blocks of life. Peptides with α-helices, long β-sheets, and long loops are usually less sensitive to D-amino acid replacements in comparison to short β-sheets.

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Fib420 , the Novel Fibrinogen Subclass: Newborn Levels Are Higher Than Adult

Blood ◽

10.1182/blood.v90.7.2609 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2609-2614 ◽

Cited By ~ 24

Author(s):

Gerd Grieninger ◽

Xianghan Lu ◽

Yan Cao ◽

Yiping Fu ◽

Bohdan J. Kudryk ◽

...

Keyword(s):

Developmental Expression ◽

Striking Difference ◽

The Novel ◽

Western Blot Assay ◽

Human Fibrinogen ◽

C Terminus ◽

Wide Range ◽

Significant Difference ◽

Adult Plasma ◽

Normal Human

Abstract Fib420 is a recently identified subclass of normal human fibrinogen in which two extended α chain isoforms (αE ) replace the common α chains, yielding a molecule (ca. 420 kD) which is larger than the more abundant 340-kD form. Evidence for preservation of this subclass throughout vertebrate evolution suggests it performs some as yet unidentified vital function. A survey was undertaken to establish the range of plasma Fib420 levels in normal, healthy adults and in placental cord (fetal) blood. For measuring Fib420 , a quantitative Western blot assay was developed using monoclonal antibody against the exon-VI encoded C-terminus of the molecule's unique αE chain. This αE chain signal was normalized to that of the β chain, common to both fibrinogen forms. Analysis of plasma samples from the adult and newborn cohorts (n = 25 each; total fibrinogen ca. 2.6 mg/mL in both) revealed a statistically significant difference, with a mean level of 100 ± 28 μg/mL in the neonate compared to 34 ± 7 μg/mL in the adult. On average, 1 out of every 100 fibrinogen molecules in adult plasma belongs to the Fib420 subclass. Unlike in the newborn, adult Fib420 levels remained the same over a wide range of total plasma fibrinogen. The striking difference observed between these two cohorts suggests a changing developmental expression of the Fib420 subclass and a homeostatic control operating in later stages of life.

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Fib420 , the Novel Fibrinogen Subclass: Newborn Levels Are Higher Than Adult

Blood ◽

10.1182/blood.v90.7.2609.2609_2609_2614 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2609-2614 ◽

Cited By ~ 2

Author(s):

Gerd Grieninger ◽

Xianghan Lu ◽

Yan Cao ◽

Yiping Fu ◽

Bohdan J. Kudryk ◽

...

Keyword(s):

Developmental Expression ◽

Striking Difference ◽

The Novel ◽

Western Blot Assay ◽

Human Fibrinogen ◽

C Terminus ◽

Wide Range ◽

Significant Difference ◽

Adult Plasma ◽

Normal Human

Fib420 is a recently identified subclass of normal human fibrinogen in which two extended α chain isoforms (αE ) replace the common α chains, yielding a molecule (ca. 420 kD) which is larger than the more abundant 340-kD form. Evidence for preservation of this subclass throughout vertebrate evolution suggests it performs some as yet unidentified vital function. A survey was undertaken to establish the range of plasma Fib420 levels in normal, healthy adults and in placental cord (fetal) blood. For measuring Fib420 , a quantitative Western blot assay was developed using monoclonal antibody against the exon-VI encoded C-terminus of the molecule's unique αE chain. This αE chain signal was normalized to that of the β chain, common to both fibrinogen forms. Analysis of plasma samples from the adult and newborn cohorts (n = 25 each; total fibrinogen ca. 2.6 mg/mL in both) revealed a statistically significant difference, with a mean level of 100 ± 28 μg/mL in the neonate compared to 34 ± 7 μg/mL in the adult. On average, 1 out of every 100 fibrinogen molecules in adult plasma belongs to the Fib420 subclass. Unlike in the newborn, adult Fib420 levels remained the same over a wide range of total plasma fibrinogen. The striking difference observed between these two cohorts suggests a changing developmental expression of the Fib420 subclass and a homeostatic control operating in later stages of life.

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Human Platelet Antigen 3 (HPA-3): Localization of the Determinant of the Alloantibody Leka (HPA-3a) to the C-Terminus of Platelet Glycoprotein IIb Heavy Chain and Contribution of O-Linked Carbohydrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651638 ◽

1993 ◽

Vol 69 (05) ◽

pp. 485-489 ◽

Cited By ~ 23

Author(s):

Isabelle Djaffar ◽

Didier Vilette ◽

Dominique Pidard ◽

Jean-Luc Wautier ◽

Jean-Philippe Rosa

Keyword(s):

Amino Acids ◽

Heavy Chain ◽

Human Platelet ◽

Platelet Glycoprotein ◽

Fibrinogen Receptor ◽

C Terminus ◽

Platelet Antigen ◽

Human Platelet Antigen ◽

Determinant Structure ◽

Polymerase Chain

SummaryThe human platelet antigen (HPA) 3 system is expressed on GPIIb, one subunit of GPIIb-IIIa, the platelet fibrinogen receptor. It was recently shown that HPA-3 was associated with an Ile843/Ser polymorphism. To investigate further HPA-3 determinant structure, we localized an HPA-3a determinant, recognized by the alloantiserum Leka, within the last 29 amino acids of GPIIbα. This region encompasses the polymorphic Ile843, which, as expected, is substituted into Ser in Leka-negative individuals, as shown by DNA sequence after polymerase chain reaction on platelet RNA. In addition, contribution of glycosylation to the determinant structure was demonstrated since the Leka antigenicity was strongly decreased after specifically removing nonterminal O-linked sugars, but not terminal sialic acids. We have thus refined the localization of an HPA-3a determinant within the last 29 amino acids, including Ile843, of GPIIb heavy chain, and shown that the Leka HPA-3a determinant is dependent, in part, upon the serine-linked carbohydrates adjacent to Ile/Ser843.

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The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽

10.3390/catal11080926 ◽

2021 ◽

Vol 11 (8) ◽

pp. 926

Author(s):

Maria C. Martins ◽

Susana F. Fernandes ◽

Bruno A. Salgueiro ◽

Jéssica C. Soares ◽

Célia V. Romão ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Reductase Activity ◽

Conserved Residues ◽

Mutant Proteins ◽

E Coli ◽

C Terminus ◽

Bona Fide ◽

Oxygen Reductase ◽

Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

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Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/150.3.977 ◽

1998 ◽

Vol 150 (3) ◽

pp. 977-986 ◽

Cited By ~ 4

Author(s):

Yangsuk Park ◽

John Hanish ◽

Arthur J Lustig

Keyword(s):

Amino Acids ◽

Active Site ◽

Fusion Proteins ◽

Initiation Site ◽

Negative Control ◽

Model System ◽

Mutant Protein ◽

Regulatory Domain ◽

C Terminus ◽

Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

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Protection of Italian ryegrass (Lolium multiflorum L.) seedlings from salinity stress following seed priming with L-methionine and casein hydrolysate

Seed Science Research ◽

10.1017/s0960258520000409 ◽

2020 ◽

pp. 1-9

Author(s):

Keum-Ah Lee ◽

Youngnam Kim ◽

Hossein Alizadeh ◽

David W.M. Leung

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Salinity Stress ◽

Lolium Multiflorum ◽

Casein Hydrolysate ◽

Seed Priming ◽

Germination Percentage ◽

Italian Ryegrass ◽

Significant Difference ◽

Protein Amino Acids

Abstract Seed priming with water (hydropriming or HP) has been shown to be beneficial for seed germination and plant growth. However, there is little information on the effects of seed priming with amino acids and casein hydrolysate (CH) compared with HP, particularly in relation to early post-germinative seedling growth under salinity stress. In this study, Italian ryegrass seeds (Lolium multiflorum L.) were primed with 1 mM of each of the 20 protein amino acids and CH (200 mg l−1) before they were germinated in 0, 60 and 90 mM NaCl in Petri dishes for 4 d in darkness. Germination percentage (GP), radicle length (RL) and peroxidase (POD) activity in the root of 4-d-old Italian ryegrass seedlings were investigated. Generally, when the seeds were germinated in 0, 60 and 90 mM NaCl, there was no significant difference in GP of seeds among various priming treatments, except that a higher GP was observed in seeds of HP treatment compared with the non-primed seeds when incubated in 60 mM NaCl. When incubated in 60 and 90 mM NaCl, seedlings from seeds primed with L-methionine or CH exhibited greater RL (greater protection against salinity stress) and higher root POD activity than those from non-primed and hydro-primed seeds. Under salinity stress, there were higher levels of malondialdehyde (MDA) in the root of 4-d-old Italian ryegrass seedlings, a marker of oxidative stress, but seed priming with CH was effective in reducing the salinity-triggered increase in MDA content. These results suggest that priming with L-methionine or CH would be better than HP for the protection of seedling root growth under salinity stress and might be associated with enhanced antioxidative defence against salinity-induced oxidative stress.

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Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus

Marine Drugs ◽

10.3390/md19040181 ◽

2021 ◽

Vol 19 (4) ◽

pp. 181

Author(s):

Kun Qiao ◽

Caiyun Jiang ◽

Min Xu ◽

Bei Chen ◽

Wenhui Qiu ◽

...

Keyword(s):

Amino Acids ◽

Von Willebrand Factor ◽

Molecular Mechanisms ◽

Protein Domain ◽

Type D ◽

Protein Toxin ◽

Factor Type ◽

Von Willebrand ◽

Willebrand Factor

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

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Cellular Membrane-Binding Ability of the C-Terminal Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope Transmembrane Protein gp41

Journal of Virology ◽

10.1128/jvi.75.20.9925-9938.2001 ◽

2001 ◽

Vol 75 (20) ◽

pp. 9925-9938 ◽

Cited By ~ 39

Author(s):

Steve S.-L. Chen ◽

Sheau-Fen Lee ◽

Chin-Tien Wang

Keyword(s):

Amino Acids ◽

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Binding ◽

Cytoplasmic Tail ◽

Cellular Membrane ◽

C Terminus ◽

Immunodeficiency Virus

ABSTRACT The amphipathic α-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli β-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the β-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated β-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.

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Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.294 ◽

1987 ◽

Vol 7 (1) ◽

pp. 294-304 ◽

Cited By ~ 30

Author(s):

D Pilgrim ◽

E T Young

Keyword(s):

Amino Acids ◽

Enzyme Activity ◽

Amino Acid ◽

Proteolytic Processing ◽

Mitochondrial Matrix ◽

Wild Type ◽

Mature Form ◽

Amino Terminal ◽

The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

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