scholarly journals Characterization of a Serine Proteinase Mediating Encystation of Acanthamoeba

2008 ◽  
Vol 7 (9) ◽  
pp. 1513-1517 ◽  
Author(s):  
Eun-Kyung Moon ◽  
Dong-Il Chung ◽  
Yeon-Chul Hong ◽  
Hyun-Hee Kong
Keyword(s):  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Tissue Destruction ◽  
Protein Fusion ◽  
Granulomatous Amoebic Encephalitis ◽  
Causative Agents ◽  
Amoebic Keratitis ◽  
Green Fluorescent ◽  
Interfering Rna

ABSTRACT Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus

2001 ◽  
Vol 69 (2) ◽  
pp. 885-896 ◽  
Author(s):  
Adhar Manna ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Virulence Determinants ◽  
Protein Fusion ◽  
Putative Gene ◽  
P2 Promoter ◽  
Green Fluorescent ◽  
Overlapping Transcripts

ABSTRACT The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g.,sar and agr). The sar locus is composed of three overlapping transcripts (sar P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp sarA gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of sar promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific column containing asar P2 promoter fragment. In this study, the putative gene, designated sarR, was identified and found to encode a 13.6-kDa protein with homology to SarA. Transcriptional and immunoblot studies revealed the sarR gene to be expressed in other staphylococcal strains. Recombinant SarR protein bound sarP1, P2, and P3 promoter fragments in gel shift and footprinting assays. A sarR mutant expressed a higher level of P1 transcript than the parent, as confirmed by promoter green fluorescent protein fusion assays. As the P1 transcript is the predominant sartranscript, we confirmed that the sarR mutant expressed more SarA than the parental strain. We thus proposed that SarR is a regulatory protein that binds to the sar promoters to down-regulate P1 transcription and the ensuing SarA protein expression.

Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi

2001 ◽  
Vol 355 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Peter C. F. CHEUNG ◽  
Laura TRINKLE-MULCAHY ◽  
Philip COHEN ◽  
John M. LUCOCQ
Keyword(s):  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Phosphate Binding ◽  
Protein Fusion ◽  
Cell Localization ◽  
Phosphate Binding Protein ◽  
Green Fluorescent ◽  
Novel Protein

We have identified a novel protein of predicted molecular mass 40kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain. The sequence of TAFF1has been deposited with the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under accession number AF311602.

scholarly journals RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro

2006 ◽  
Vol 80 (20) ◽  
pp. 10055-10063 ◽  
Author(s):  
Adrian Valli ◽  
Ana Montserrat Martín-Hernández ◽  
Juan José López-Moya ◽  
Juan Antonio García
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Plum Pox Virus ◽  
Coding Region ◽  
Long Distance ◽  
Systemic Spread ◽  
The Family ◽  
Silencing Suppression ◽  
Green Fluorescent

ABSTRACT The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

Sign in / Sign up

Export Citation Format

Share Document