Evaluating the potential of non-immunosuppressive cyclosporin analogs for targeting Toxoplasma gondii cyclophilin: Insights from structural studies.

Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets.

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (HS) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections.

Conformational and dynamic properties of the KH1 domain of FMRP and its fragile X syndrome linked G266E variant.

The Fragile X messenger ribonucleoprotein (FMRP) is a multi-domain protein involved in interactions with various macromolecules, including proteins and coding/non-coding RNAs. The three KH domains (KH0, KH1 and KH2) within FMRP are recognized for their roles in mRNA binding. In the context of Fragile X syndrome (FXS), over-and-above CGG triplet repeats expansion, three specific point mutations have been identified, each affecting one of the three KH domains (KH0, KH1, and KH2) resulting in the expression of non-functional FMRP.

Biochemical and structural impact of two novel missense mutations in cystathionine β-synthase gene associated with homocystinuria.

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems.

Hydrogen sulfide production does not affect antibiotic resistance in .

Hydrogen sulfide (HS) has been proposed to protect bacteria from antibiotics, pointing to HS-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with mutants producing altered HS levels demonstrate that HS does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of cystic fibrosis isolates argue against the protective role of HS from antibiotic activity during chronic lung infection.

Role of myristoylation in modulating PCaP1 interaction with calmodulin.

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca-CaM interaction and the functional impact of myristoylation in the complex formation and Ca sensitivity of CaM remained to be elucidated.

Key substrate recognition residues in the active site of cystathionine beta-synthase from Toxoplasma gondii.

Cystathionine β-synthase (CBS) catalyzes the condensation of l-serine and l-homocysteine to give l-cystathionine in the transsulfuration pathway. Recently, a few O-acetylserine (l-OAS)-dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l-OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l-serine and l-OAS to form l-cystathionine.

Structural basis of the inhibition of cystathionine γ-lyase from Toxoplasma gondii by propargylglycine and cysteine.

Cystathionine γ-lyase (CGL) is a PLP-dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL-catalyzed process consists of an α,γ-elimination reaction that breaks down cystathionine into cysteine, α-ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H S).

Conformational Plasticity of Centrin 1 from in Binding to the Centrosomal Protein SFI1.

Centrins are calcium (Ca)-binding proteins that are involved in many cellular functions including centrosome regulation. A known cellular target of centrins is SFI1, a large centrosomal protein containing multiple repeats that represent centrin-binding motifs. Recently, a protein homologous to yeast and mammalian SFI1, denominated TgSFI1, which shares SFI1-repeat organization, was shown to colocalize at centrosomes with centrin 1 from (TgCEN1).

Insights into Domain Organization and Regulatory Mechanism of Cystathionine Beta-Synthase from .

Cystathionine beta-synthase (CBS) is a key regulator of homocysteine metabolism. Although eukaryotic CBS have a similar domain architecture with a catalytic core and a C-terminal Bateman module, their regulation varies widely across phyla. In human CBS (HsCBS), the C-terminus has an autoinhibitory effect by acting as a cap that avoids the entry of substrates into the catalytic site.

Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides.

Calcium (Ca) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca signals are decoded into defined plant adaptive responses by different Ca sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown.

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition.

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca) binding site that is non-functional, thus displaying large variance in Ca sensing abilities that could support their functional versatility.

Structural insight into the unique conformation of cystathionine β-synthase from .

Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is , the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL).

The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii.

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble.

Structural Insights into the Heme Pocket and Oligomeric State of Non-Symbiotic Hemoglobins from .

Non-symbiotic hemoglobins AHb1 and AHb2 from are hexacoordinate heme-proteins that likely have different biological roles, in view of diverse tissue localization, expression pattern, and ligand binding properties. Herein, we expand upon previous biophysical studies on these isoforms, focusing on their oligomeric states and circular dichroism (CD) characteristics. We found that AHb1 exists in solution in a concentration-dependent monomer-dimer equilibrium, while AHb2 is present only as a monomer.

Cystathionine β-synthase is involved in cysteine biosynthesis and HS generation in Toxoplasma gondii.

Cystathionine β-synthase (CBS) catalyzes the condensation of serine and homocysteine to water and cystathionine, which is then hydrolyzed to cysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CGL) in the reverse transsulfuration pathway. The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, includes both CBS and CGL enzymes. We have recently reported that the putative T.

Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from .

Centrins are calcium (Ca)-binding proteins that have been implicated in several regulatory functions. In the protozoan parasite , the causative agent of toxoplasmosis, three isoforms of centrin have been identified. While increasing information is now available that links the function of centrins with defined parasite biological processes, knowledge is still limited on the metal-binding and structural properties of these proteins.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana.

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown.

A Method for Metal/Protein Stoichiometry Determination Using Thin-Film Energy Dispersive X-ray Fluorescence Spectroscopy.

A convenient approach is proposed for the quantitation of elemental cofactors in proteins and the determination of metal/protein stoichiometry, on the basis of energy dispersive X-ray fluorescence spectroscopy (EDXRF). The analysis of proteins containing the metals Cu, Fe, Zn, and Ca and also the nonmetallic element P is shown as a demonstration of the generality of the method. In general, the reported method gives limit of detection (LOD) and limit of quantification (LOQ) values in the low ppm range and requires only a few microliters of protein sample at micromolar concentrations.

Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana.

Plants contain a large family of so-called calmodulin-like proteins (CMLs) which differ from canonical calmodulin in that they show greater variability in sequence, length, and number of EF-hand domains. The presence of this extended CML family has raised questions regarding the role of these proteins: are they functionally redundant or do they play specific functions in physiological plant processes? To answer these questions, comprehensive biochemical and structural information on CML proteins is fundamental. Among the 50 CMLs from Arabidopsis thaliana, herein we described the ability of CML7 to bind metal ions focusing on the Ca and Mg sensing properties, as well as on metal-induced conformational changes.

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from .

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of .

Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses.

Calcium (Ca) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes.

Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective.

Ca ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca-binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca sensors in plants.

Phosphorylation of pyridoxal 5'-phosphate enzymes: an intriguing and neglected topic.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes catalyze a wide range of reactions of amino acids and amines, with the exception of glycogen phosphorylase which exhibits peculiar both substrate preference and chemical mechanism. They represent about 4% of the gene products in eukaryotic cells. Although structure-function investigations regarding these enzymes are copious, their regulation by post-translational modifications is largely unknown.