A major virulence trait of
(
) is its ability to enter a dormant state within its human host. Since cell division is intimately linked to metabolic shut down, understanding the mechanism of septum ...formation and its integration with other events in the division pathway is likely to offer clues to the molecular basis of dormancy. The
genome lacks obvious homologues of several conserved cell division proteins, and this study was aimed at identifying and functionally characterising mycobacterial homologues of the
septum site specification protein MinD (
MinD). Sequence homology based analyses suggested that the genomes of both
and the saprophyte
(
) encode two putative
MinD homologues
and
. Of these,
were found to be the true homologues, through complementation of the
mutant HL1, overexpression studies, and structural comparisons.
and
fully complemented the mini-cell phenotype of HL1, and over-expression of
in
led to cell elongation and a drastic decrease in c.f.u. counts, indicating its essentiality in cell-division. MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif. Interaction of Rv1708 with the chromosome associated proteins ScpA and ParB, implied a link between its septum formation role, and chromosome segregation. Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c. In summary we identify Rv1708 and MSMEG_3743 to be homologues of
MinD, adding a critical missing piece to the mycobacterial cell division puzzle.
The angiotensin II receptors AT
R and AT
R serve as key components of the renin-angiotensin-aldosterone system. AT
R has a central role in the regulation of blood pressure, but the function of AT
R ...is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT
R bound to an AT
R-selective ligand and to an AT
R/AT
R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
Snake venoms are cocktails of enzymes and non‐enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l‐amino ...acid oxidases, serine proteinases, metalloproteinases and phospholipases A2. Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure‐based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non‐enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor–enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.
Snake venoms are cocktails of enzymes and non‐enzymatic proteins. Here, we describe the structures of the common snake venom enzymes, namely, acetylcholinesterase, L‐amino acid oxidase, phospholipase A2, serine proteinase, and metalloproteinase, and their complexes. We discuss the contribution of these structures in understanding the mechanisms of catalysis and inhibition as well as in the structure‐based design of new, potent inhibitors
Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial ...and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.
Lactoferrin (LF) is an iron-binding glycoprotein released from mucous secreting cells and neutrophils. LF can be used in a broad range of eye diseases related to the retina, cornea, and optic nerve. ...The retina is particularly affected by oxidative stress inside the photoreceptor being constantly exposed to light which induces accumulation of reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE) causing damage to photoreceptor recycling. Retinitis pigmentosa (RP) and macular degeneration are inherited retinopathies that consist of different disease-causing genes, that cause mutations with highly varied clinical consequences. Age-related macular degeneration is a chronic disease of the retina and one of the major causes of sight loss. This review provides an application of lactoferrin and LF-based nano-formulations or nanoparticles in the field of retinal diseases or corneal diseases such as retinitis pigmentosa, retinoblastoma, age-related macular degeneration (AMD), keratoconus and uveitis. Several studies have found that lactoferrin's antibacterial activity is not limited to its iron sequestration, but also its ability as a nanoparticle that acts as a carrier to deliver drugs by crossing the blood-retina barrier (BRB) and its involvement in cell cycle control, which is not possible by many transferrin proteins.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human ...diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer's interfaces reduce IL-1β signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism.
The cooperative assembly of FtsZ, the prokaryotic homologue of tubulin, plays an essential role in cell division. FtsZ is a potential drug target, as illustrated by the small-molecule cell-cycle ...inhibitor and antibacterial agent PC190723 that targets FtsZ. We demonstrate that PC190723 negatively modulates Staphylococcus aureus FtsZ polymerization cooperativity as reflected in polymerization at lower concentrations without a defined critical concentration. The crystal structure of the S. aureus FtsZ-PC190723 complex shows a domain movement that would stabilize the FtsZ protofilament over the monomeric state, with the conformational change mediated from the GTP-binding site to the C-terminal domain via helix 7. Together, the results reveal the molecular mechanism of FtsZ modulation by PC190723 and a conformational switch to the high-affinity state that enables polymer assembly.
The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas ...aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear.
The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK).
The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies.
NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF.
The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.
The efficiency of heterologous protein production in Escherichia coli (E. coli) can be diminished by biased codon usage. Approaches normally used to overcome this problem include targeted mutagenesis ...to remove rare codons or the addition of rare codon tRNAs in specific cell lines. Recently, improvements in technology have enabled cost-effective production of synthetic genes, making this a feasible alternative. To explore this option, the expression patterns in E. coli of 30 human short-chain dehydrogenase/reductase genes (SDRs) were analyzed in three independent experiments, comparing the native and synthetic (codon-optimized) versions of each gene. The constructs were prepared in a pET-derived vector that appends an N-terminal polyhistidine tag to the protein; expression was induced using IPTG and soluble proteins were isolated by Ni–NTA metal-affinity chromatography. Expression of the native and synthetic gene constructs was compared in two isogenic bacterial strains, one of which contained a plasmid (pRARE2) that carries seven tRNAs recognizing rare codons. Although we found some degree of variability between experiments, in normal E. coli synthetic genes could be expressed and purified more readily than the native version. In only one case was native gene expression better. Importantly, in most but not all cases, expression of the native genes in combination with rare codon tRNAs mimicked the behavior of the synthetic genes in the native strain. The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.
The problem of antibiotic resistance has prompted researchers around the globe to search for new antimicrobial agents. Antimicrobial proteins and peptides are naturally secreted by almost all the ...living organisms to fight infections and can be safer alternatives to chemical antibiotics. Lactoferrin (LF) is a known antimicrobial protein present in all body secretions. In this study, LF was digested by trypsin, and the resulting hydrolysates were studied with respect to their antimicrobial properties. Among the hydrolysates, a 21-kDa basic fragment of LF (termed lactosmart) showed promise as a new potent antimicrobial agent. The antimicrobial studies were performed on various microorganisms including
Shigella flexneri
,
Pseudomonas aeruginosa
,
Staphylococcus aureus
, and
Escherichia coli
as well as fungal pathogens such as
Candida albicans
,
Candida tropicalis
, and
Candida glabrata
. In addition, the lipopolysaccharide (LPS)-binding properties of lactosmart were studied using surface plasmon resonance technique
in vitro
, along with docking of LPS and molecular dynamics (MD) simulation studies. The results showed that lactosmart had better inhibitory effects against pathogenic microorganisms compared to LF. The results of docking and MD simulation studies further validated the tighter binding of LPS to lactosmart compared to LF. The two LPS-binding sites have been characterized structurally in detail. Through these studies, it has been demonstrated that in native LF, only one LPS-binding site remains exposed due to its location being on the surface of the molecule. However, due to the generation of the lactosmart molecule, the second LPS-binding site gets exposed too. Since LPS is an essential and conserved part of the bacterial cell wall, the pro-inflammatory response in the human body caused by LPS can be targeted using the newly identified lactosmart. These findings highlight the immense potential of lactosmart in comparison to native LF in antimicrobial defense. We propose that lactosmart can be further developed as an antibacterial, antifungal, and antibiofilm agent.
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