Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.

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Functional characterization of heat-shock protein 90 fromOryza sativaand crystal structure of its N-terminal domain

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15006639 ◽

2015 ◽

Vol 71 (6) ◽

pp. 688-696 ◽

Cited By ~ 1

Author(s):

Swetha Raman ◽

Kaza Suguna

Keyword(s):

Crystal Structure ◽

Heat Shock ◽

Heat Shock Protein ◽

Biochemical Characterization ◽

Functional Characterization ◽

Cell Signalling ◽

Physiological Role ◽

Heat Shock Protein 90 ◽

Terminal Domain

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 fromDictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

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Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽

10.3390/plants10030524 ◽

2021 ◽

Vol 10 (3) ◽

pp. 524

Author(s):

Bingqi Wu ◽

Zhiting Chen ◽

Xiaohui Xu ◽

Ronghua Chen ◽

Siwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Heterologous Gene Expression ◽

Expression System ◽

Functional Characterization ◽

Transient Gene Expression ◽

High Mobility ◽

Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

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Crystal structure and functional characterization of a cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp.

PLoS ONE ◽

10.1371/journal.pone.0206260 ◽

2018 ◽

Vol 13 (10) ◽

pp. e0206260 ◽

Cited By ~ 5

Author(s):

Sun-Ha Park ◽

Wanki Yoo ◽

Chang Woo Lee ◽

Chang Sook Jeong ◽

Seung Chul Shin ◽

...

Keyword(s):

Crystal Structure ◽

Functional Characterization ◽

Acetyl Xylan Esterase ◽

Soil Microbe ◽

Paenibacillus Sp ◽

Cold Active

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Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

Physiological Genomics ◽

10.1152/physiolgenomics.2001.5.3.137 ◽

2001 ◽

Vol 5 (3) ◽

pp. 137-145 ◽

Cited By ~ 56

Author(s):

CLAUDIA R. VIANNA ◽

THILO HAGEN ◽

CHEN-YU ZHANG ◽

ERIC BACHMAN ◽

OLIVIER BOSS ◽

...

Keyword(s):

Uncoupling Protein ◽

Expression System ◽

Functional Characterization ◽

Pectoral Muscle ◽

Mitochondrial Fraction ◽

Mrna Levels ◽

Coding Region ◽

Reading Frame ◽

Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

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Functional Characterization of Cholinergic Receptors in Melanoma Cells

Cancers ◽

10.3390/cancers12113141 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3141

Author(s):

Anna Maria Lucianò ◽

Ada Maria Tata

Keyword(s):

Nervous System ◽

Acetylcholine Receptors ◽

Epithelial Mesenchymal Transition ◽

Functional Characterization ◽

Physiological Role ◽

Melanoma Cells ◽

Cholinergic Receptors ◽

Mesenchymal Transition ◽

Relevant Role

In the last two decades, the scientific community has come to terms with the importance of non-neural acetylcholine in light of its multiple biological and pathological functions within and outside the nervous system. Apart from its well-known physiological role both in the central and peripheral nervous systems, in the autonomic nervous system, and in the neuromuscular junction, the expression of the acetylcholine receptors has been detected in different peripheral organs. This evidence has contributed to highlight new roles for acetylcholine in various biological processes, (e.g., cell viability, proliferation, differentiation, migration, secretion). In addition, growing evidence in recent years has also demonstrated new roles for acetylcholine and its receptors in cancer, where they are involved in the modulation of cell proliferation, apoptosis, angiogenesis, and epithelial mesenchymal transition. In this review, we describe the functional characterization of acetylcholine receptors in different tumor types, placing attention on melanoma. The latest set of data accessible through literature, albeit limited, highlights how cholinergic receptors both of muscarinic and nicotinic type can play a relevant role in the migratory processes of melanoma cells, suggesting their possible involvement in invasion and metastasis.

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High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme

Biochemical Journal ◽

10.1042/bj2940069 ◽

1993 ◽

Vol 294 (1) ◽

pp. 69-77 ◽

Cited By ~ 27

Author(s):

C Wang ◽

M R Lee

Keyword(s):

Escherichia Coli ◽

Dissociation Constants ◽

Expression System ◽

Deae Cellulose ◽

Basic Amino Acid ◽

Coated Vesicle ◽

Amino Acid Residues ◽

High Level ◽

Binding Component

We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.

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Functional Characterization of a Na+-Coupled Dicarboxylate Carrier Protein from Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.187.15.5189-5194.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5189-5194 ◽

Cited By ~ 21

Author(s):

Jason A. Hall ◽

Ana M. Pajor

Keyword(s):

Staphylococcus Aureus ◽

Structural Analysis ◽

Transport Properties ◽

Sequence Homology ◽

Cytoplasmic Membrane ◽

Functional Characterization ◽

Carrier Protein ◽

Dependent Manner ◽

Reaction Sequence

ABSTRACT We have cloned and functionally characterized a Na+-coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na+ symporter (DASS) family and shares significant sequence homology with the mammalian Na+/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na+-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na+ (K 0.5 of 2.7 mM) binding before dicarboxylate (Km of 4.5 μM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.

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