Prokaryotic Proteins Research Articles

Improvement of extracellular bacterial protein production in Pichia pastoris by co-expression of endoplasmic reticulum residing GroEL–GroES

Pichia pastoris is an established host system for heterologous protein expression. However, the potential productivity of this system can be limited. In this study, the Escherichia coli chaperones (GroES-GroEL) were expressed from the PGAP promoter and targeted to the secretory pathway through the endoplasmic reticulum (ER). The ability of the ER targeted chaperones to improve production of bacterial protein in P.pastoris was evaluated. The chaperones tagged with α-factor secretion- and ER retention-signal sequences were co-expressed with either extracellularly secreted phytase or intracellular d-phenylglycine aminotransferase (D-PhgAT) enzymes. The ER residing GroEL-GroES successfully increased the levels of active phytase extracellularly, 1.5-2.3-fold higher than the phytase expression alone, but did not enhance the formation of active, intracellular D-PhgAT. These results indicate that the chaperones have the potential to enhance production of active enzymes when present in the same trafficking pathway. This is the first report on the improvement of extracellular bacterial protein production through co-expression with ER residing bacterial chaperones in the Pichia system. The modified P.pastoris expression system may be beneficial for extracellular expression of other prokaryotic proteins.

Nucleic Acid-Binding Assay of Argonaute Protein Using Fluorescence Polarization.

Nucleic acid binding by the Argonaute protein is an important trigger step in the Argonaute-dependent gene silencing system. We established an in vitro method to detect the nucleic acid binding activity of the Argonaute protein by fluorescence polarization. In this chapter, we will describe the expression and purification of the prokaryotic (Rhodobacter sphaeroides) Argonaute protein, and the nucleic acid-binding analysis using a Fluorescence Polarization System (Beacon 2000).

Energy coupling mechanisms of AcrB-like RND transporters

Prokaryotic AcrB-like proteins belong to a family of transporters of the RND superfamily, and as main contributing factor to multidrug resistance pose a tremendous threat to future human health. A unique feature of AcrB transporters is the presence of two separate domains responsible for carrying substrate and generating energy. Significant progress has been made in elucidating the three-dimensional structures of the homo-trimer complexes of AcrB-like transporters, and a three-step functional rotation was identified for this class of transporters. However, the detailed mechanisms for the transduction of the substrate binding signal, as well as the energy coupling processes between the functionally distinct domains remain to be established. Here, we propose a model for the interdomain communication in AcrB that explains how the substrate binding signal from the substrate-carrier domain triggers protonation in the transmembrane domain. Our model further provides a plausible mechanism that explains how protonation induces conformational changes in the substrate-carrier domain. We summarize the thermodynamic principles that govern the functional cycle of the AcrB trimer complex.

The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation

Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

Prokaryotic and Highly-Repetitive WD40 Proteins: A Systematic Study

As an ancient protein family, the WD40 repeat proteins often play essential roles in fundamental cellular processes in eukaryotes. Although investigations of eukaryotic WD40 proteins have been frequently reported, prokaryotic ones remain largely uncharacterized. In this paper, we report a systematic analysis of prokaryotic WD40 proteins and detailed comparisons with eukaryotic ones. About 4,000 prokaryotic WD40 proteins have been identified, accounting for 6.5% of all WD40s. While their abundances are less than 0.1% in most prokaryotes, they are enriched in certain species from Cyanobacteria and Planctomycetes, and participate in various functions such as prokaryotic signal transduction and nutrient synthesis. Comparisons show that a higher proportion of prokaryotic WD40s tend to contain multiple WD40 domains and a large number of hydrogen bond networks. The observation that prokaryotic WD40 proteins tend to show high internal sequence identity suggests that a substantial proportion of them (~20%) should be formed by recent or young repeat duplication events. Further studies demonstrate that the very young WD40 proteins, i.e., Highly-Repetitive WD40s, should be of higher stability. Our results have presented a catalogue of prokaryotic WD40 proteins, and have shed light on their evolutionary origins.

Virulence protein VirD5 of Agrobacterium tumefaciens binds to kinetochores in host cells via an interaction with Spt4

The bacterium Agrobacterium tumefaciens causes crown gall tumor formation in plants. During infection the bacteria translocate an oncogenic piece of DNA (transferred DNA, T-DNA) into plant cells at the infection site. A number of virulence proteins are cotransported into host cells concomitantly with the T-DNA to effectuate transformation. Using yeast as a model host, we find that one of these proteins, VirD5, localizes to the centromeres/kinetochores in the nucleus of the host cells by its interaction with the conserved protein Spt4. VirD5 promotes chromosomal instability as seen by the high-frequency loss of a minichromosome in yeast. By using both yeast and plant cells with a chromosome that was specifically marked by a lacO repeat, chromosome segregation errors and the appearance of aneuploid cells due to the presence of VirD5 could be visualized in vivo. Thus, VirD5 is a prokaryotic virulence protein that interferes with mitosis.

Prokaryotic Argonaute proteins: novel genome-editing tools?

Argonaute proteins constitute a highly diverse family of nucleic acid-guided proteins. They were first discovered in eukaryotes as key proteins in RNA interference systems, but homologous prokaryotic Argonaute proteins (pAgos) have also been found in archaea and bacteria. In this Progress article, we focus on long pAgo variants, a class of pAgos that are involved in nucleic acid-guided host defence against invading nucleic acids, and discuss the potential of pAgos in genome editing.

2,6-Difluorobenzamide Inhibitors of Bacterial Cell Division Protein FtsZ: Design, Synthesis, and Structure-Activity Relationships.

A wide variety of drug-resistant microorganisms are continuously emerging, restricting the therapeutic options for common bacterial infections. Antimicrobial agents that were originally potent are now no longer helpful, due to their weak or null activity toward these antibiotic-resistant bacteria. In addition, none of the recently approved antibiotics affect innovative targets, resulting in a need for novel drugs with innovative antibacterial mechanisms of action. The essential cell division protein filamentous temperature-sensitive Z (FtsZ) has emerged as a possible target, thanks to its ubiquitous expression and its homology to eukaryotic β-tubulin. In the latest years, several compounds were shown to interact with this prokaryotic protein and selectively inhibit bacterial cell division. Recently, our research group developed interesting derivatives displaying good antibacterial activities against methicillin-resistant Staphylococcus aureus, as well as vancomycin-resistant Enterococcus faecalis and Mycobacterium tuberculosis. The aim of the present study was to summarize the structure-activity relationships of differently substituted heterocycles, linked by a methylenoxy bridge to the 2,6-difluorobenzamide, and to validate FtsZ as the real target of this class of antimicrobials.

Plant E3 ligases ubiquitinate Escherichia coli σ32in vitro

Ubiquitin-like proteins (UBLs) are extremely well-conserved among eukaryotes and prokaryotes allowing interactions between proteins from different organisms. For example, the prokaryotic ubiquitin-like proteins (Pups) and the Proteasome accessory factor A (PafA) of Mycobacterium tuberculosis are sufficient to pupylate at least 51 Escherichia coli proteins. This work shows that the plant E3 ligases BnTR1 and AT1G02860 can ubiquitinate E. coli σ32, but not Hsp70 DnaK in vitro. Molecular biology and biochemical studies confirm that the RING finger domain of BnTR1 and AT1G02860 is essential for their function. These results suggest that the substrates of plant E3 ligases can be prokaryotic protein and therefore the plant ubiquitylation system may have evolved from prokaryote.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter-repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

Antibody Response to Human Extracellular HER2 Subdomain Proteins in Mice.

In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.

DNA recognition by an RNA-guided bacterial Argonaute.

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.

Distribution and evolution of stable single α-helices (SAH domains) in myosin motor proteins.

Stable single-alpha helices (SAHs) are versatile structural elements in many prokaryotic and eukaryotic proteins acting as semi-flexible linkers and constant force springs. This way SAH-domains function as part of the lever of many different myosins. Canonical myosin levers consist of one or several IQ-motifs to which light chains such as calmodulin bind. SAH-domains provide flexibility in length and stiffness to the myosin levers, and may be particularly suited for myosins working in crowded cellular environments. Although the function of the SAH-domains in human class-6 and class-10 myosins has well been characterised, the distribution of the SAH-domain in all myosin subfamilies and across the eukaryotic tree of life remained elusive. Here, we analysed the largest available myosin sequence dataset consisting of 7919 manually annotated myosin sequences from 938 species representing all major eukaryotic branches using the SAH-prediction algorithm of Waggawagga, a recently developed tool for the identification of SAH-domains. With this approach we identified SAH-domains in more than one third of the supposed 79 myosin subfamilies. Depending on the myosin class, the presence of SAH-domains can range from a few to almost all class members indicating complex patterns of independent and taxon-specific SAH-domain gain and loss.

Establishment of Double Antibody Sandwich ELISA for the Determination of Human Soluble VE-Cadherin in Human Plasma and Its Application

To establish double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human soluble VE-cadherin in human plasma and to investigate its value in clinical use. The monoclonal antibodies against human VE-cadherin were prepared from BALB/c mice immunized with prokaryotic expression recombinant proteins, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human VE-cadherin was established by using HRP-labeled McAb as a detection antibody and a capture antibody. The methodology performance was evaluated. The plasma concentrations of VE-cadherin in 28 healthy subjects and 60 patients with cancer were determined. The double antibody sandwich ELISA for the determination of human VE-cadherin was established by selecting the combination of double antibodies. The detection limit was 24.7 pg/ml, the coefficients of variation for inner-batch and inter-batch were 4.1%-7.7% and 8.7%-10.8% respectively. The average recovery was 96.7%. The plasma level of soluble VE-cadherin in normal controls was 262.1±11.75 pg/ml. The plasma level of soluble VE-cadherin was 173.9±17.98 pg/ml in 24 patients with leukemia, 311.7±25.24 pg/ml in 14 patients with stomach cancer, 206.8±25.01 pg/ml in 11 patients with lung cancer, and 310.7±11.82 pg/ml in others patients(9 patients with breast cancer, 1 patients with gliomas, 1 patients with liver cancer). The developed ELISA kit has better sensitivity and specificity, and can be used in detection of human soluble VE-cadherin in human plasma, therefore, it can provide a new mathod for diagnosis of cancer patients.

Prion-like characteristics of the bacterial protein Microcin E492

Microcin E492 (Mcc) is a pore-forming bacteriotoxin. Mcc activity is inhibited at the stationary phase by formation of amyloid-like aggregates in the culture. Here we report that, in a similar manner as prions, Mcc naturally exists as two conformers: a β-sheet-rich, protease-resistant, aggregated, inactive form (Mccia), and a soluble, protease-sensitive, active form (Mcca). The exogenous addition of culture medium containing Mccia or purified in vitro-generated Mccia into the culture induces the rapid and efficient conversion of Mcca into Mccia, which is maintained indefinitely after passaging, changing the bacterial phenotype. Mccia prion-like activity is conformation-dependent and could be reduced by immunodepleting Mccia. Interestingly, an internal region of Mcc shares sequence similarity with the central domain of the prion protein, which is key to the formation of mammalian prions. A synthetic peptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducing the conversion of Mcca into Mcciain vivo, suggesting that this region corresponds to the prion domain of Mcc. Our findings suggest that Mcc is the first prokaryotic protein with prion properties which harnesses prion-like transmission to regulate protein function, suggesting that propagation of biological information using a prion-based conformational switch is an evolutionary conserved mechanism.

Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii.

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochrome b558 composed of NOX2 and p22phox, the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22phox liposomes was estimated to be around 700 nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47phox, p67phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22phox liposomes for 8 h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments.

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

Prokaryotic Argonaute Uses an All-in-One Mechanism to Provide Host Defense

In this issue, Swarts etal. (2017) discover that prokaryotic Argonaute proteins generate their own DNA guide strands through a newly characterized, unguided "chopping" activity. Double-stranded chopping products are selectively loaded onto Argonaute, enabling DNA-guided defense against foreign DNA.

Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why invivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.