1,721,021 research outputs found
Recognition and binding of apocytochrome c to P. aeruginosa CcmI, a component of cytochrome c maturation machinery.
No full textThe biogenesis of c-type cytochromes (Cytc) is a process that in Gram-negative bacteria demands the coordinated action of different periplasmic proteins (CcmA-I), whose specific roles are still being investigated. Activities of Ccm proteins span from the chaperoning of heme b in the periplasm to the specific reduction of oxidized apocytochrome (apoCyt) cysteine residues and to chaperoning and recognition of the unfolded apoCyt before covalent attachment of the heme to the cysteine thiols can occur. We present here the functional characterization of the periplasmic domain of CcmI from the pathogen Pseudomonas aeruginosa (Pa-CcmI*). Pa-CcmI* is composed of a TPR domain and a peculiar C-terminal domain. Pa-CcmI* fulfills both the ability to recognize and bind to P. aeruginosa apo-cytochrome c551 (Pa-apoCyt) and a chaperoning activity towards unfolded proteins, as it prevents citrate synthase aggregation in a concentration-dependent manner. Equilibrium and kinetic experiments with Pa-CcmI
Molecules that target nucleophosmin for cancer treatment: an update
Full text linkNucleophosmin is a highly and ubiquitously expressed protein, mainly localized in nucleoli but able to shuttle between nucleus and cytoplasm. Nucleophosmin plays crucial roles in ribosome maturation and export, centrosome duplication, cell cycle progression, histone assembly and response to a variety of stress stimuli. Much interest in this protein has arisen in the past ten years, since the discovery of heterozygous mutations in the terminal exon of the NPM1 gene, which are the most frequent genetic alteration in acute myeloid leukemia. Nucleophosmin is also frequently overexpressed in solid tumours and, in many cases, its overexpression correlates with mitotic index and metastatization. Therefore it is considered as a promising target for the treatment of both haematologic and solid malignancies. NPM1 targeting molecules may suppress different functions of the protein, interfere with its subcellular localization, with its oligomerization properties or drive its degradation. In the recent years, several such molecules have been described and here we review what is currently known about them, their interaction with nucleophosmin and the mechanistic basis of their toxicity. Collectively, these molecules exemplify a number of different strategies that can be adopted to target nucleophosmin and we summarize them at the end of the review
The folding pathway of a functionally competent C-terminal domain of nucleophosmin: Protein stability and denatured state residual structure.
No full textNucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins
Nucleophosmin acute myeloid leukemia mutant is unable to bind G-quadruplex sequences at ribosomal DNA in vitro.
No full textNucleophosmin (NPM1) is an abundant phospho-protein that plays a key role in ribosome biogenesis. NPM1 binds nucleic acids and has intrinsic RNAase and chaperone activities. Although mainly localized at nucleoli, NPM1 continuously shuttles between the nucleus and the cytoplasm to fullfill its functions.
The gene encoding NPM1 is mutated in 50–60% of normal kariotype acute myeloid leukemia (AML) patients. These mutants are stably localized in the cytoplasm of leukemic cells due to the critical changes at the NPM1 C-terminus domain: i) loss of tryptophan residues 288 and 290 (or 290 alone), which destabilizes the C-terminal domain; ii) generation of a new Nuclear Export Signal (NES) motif. Both alterations are critical for perturbing NPM1 mutant traffic and for the underlying aberrant accumulation of NPM1 in the cytoplasm of leukemic cells.
We investigated the structural determinants of NPM1 nucleolar localization in vitro by means of Surface Plasmon Resonance (SPR). We showed that a domain encompassing the last 70 residues of the protein (NPM1-C70) binds with high affinity oligonucleotide sequences with G-quadruplex structure found at the non-coding strand of ribosomal DNA. Importantly, the most common leukemic NPM1 variant C-terminal domain, loses this DNA binding activity being completely unfolded. Since mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex binding activity, we suggest that NPM1 nucleolar localization depends on its interaction with G-quadruplex regions at rDNA, which is impaired by AML-associated mutations
Polygalacturonase-inhibiting proteins: players in plant innate immunity?
No full textPolygalacturonase-inhibiting proteins (PGIPs) are extracellular
leucine-rich repeat (LRR) proteins that recognize
and inhibit fungal polygalacturonases (PGs).
The PG–PGIP interaction favours the accumulation of
elicitor-active oligogalacturonides and causes the activation
of defence responses. Small gene families encode
PGIP isoforms that differ in affinity and specificity for
PGs secreted by different pathogens. The consensus
motif within the LRR structure of PGIPs is the same as
that of the extracellular receptors of the plant innate
immune system. Structural and functional evidence
suggest that PGIPs are versatile proteins involved in
innate immunity and that they are capable of recognizing
different surface motifs of functionally related but
structurally variable PGs
Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides
No full textCalcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants. (C) 2021 Elsevier Inc. All rights reserved
NO-sensing in Pseudomonas aeruginosa: Structure of the transcriptional regulator DNR
No full textAll denitrifying bacteria can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels, controlling the expression of the denitrification gene clusters by redox signaling, mainly through transcriptional regulators belonging either to the DNR (dissimilative nitrate respiration regulator) or to the NnrR (nitrite and nitric oxide reductase regulator) subgroups of the FNR (fumarate and nitrate reductase regulatory protein)–CRP (cAMP receptor protein) superfamily. The NO dependence of the transcriptional activity of promoters regulated by these transcription factors has suggested that they may act as NO sensors in vivo. Despite great interest in the regulation of denitrification, which in Pseudomonas aeruginosa is strictly related to virulence, functional and structural characterization of these NO sensors is still lacking. Here we present the three-dimensional structure of the sensor domain of the DNR from P. aeruginosa at 2.1 Å resolution. This is the first structure of a putative NO-sensing bacterial transcriptional regulator and reveals the presence of a large hydrophobic cavity that may be the cofactor binding site. Parallel spectroscopic evidence indicates that apo-DNR binds heme in vitro and that the heme-bound form reacts with carbon monoxide and NO, thus supporting the hypothesis that NO sensing involves gas binding to the ferrous heme. Preliminary experiments indicate that heterologous expression of the heme-containing DNR yields a protein able to bind DNA in vitro
Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana
No full textPlants 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 Ca2+ and Mg2+ sensing properties, as well as on metal-induced conformational changes. Circular dichroism and nuclear magnetic resonance (NMR) studies indicated that both Ca2+ and Mg2+ stabilize CML7, as reflected in conformational rearrangements in secondary and tertiary structure and in increases in thermal stability of the protein. However, the conformational changes that binding induces differ between the two metal ions, and only Ca2+ binding controls a structural transition that leads to hydrophobic exposure, as suggested by 8-anilino-1-naphthalenesulfonic acid fluorescence. Isothermal titration calorimetry data coupled with NMR experiments revealed the presence of two high affinity Ca2+-binding sites in the C-lobe of CML7 and two weaker sites in the N-lobe. The paired nature of these CML7 EF-hands enables them to bind Ca2+ with positive cooperativity within each globular domain. Our results clearly place CML7 in the category of Ca2+ sensors. Along with this, the protein can bind to a model target peptide (melittin) in a Ca2+-dependent manner
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