Deciphering the folding transition state structure and denatured state properties of Nucleophosmin C-terminal domain

Nucleophosmin (NPM1), one of the most abundant nucleolar proteins, is a frequent target of oncogenic mutations in acute myeloid leukaemia (AML). Mutation-induced changes at the C-terminal domain of NPM1 (Cter-NPM1) compromise its stability and cause the aberrant translocation of NPM1 to the cytosol. Hence, this protein represents a suitable candidate to investigate the relations between folding and disease. Since Cter-NPM1 folds via a compact denatured state, stabilization of the folded state of the mutated variants demands detailed structural information on both the native and denatured states. Here, we present the characterization of the complete folding pathway of Cter-NPM1 and provide molecular details for both the transition and the denatured states. The structure of the transition state was assessed by Φ-value analysis, whereas residual structure in the denatured state was mapped by evaluating the effect of mutations as modulated by conditions promoting denatured state compaction. Data reveal that folding of Cter-NPM1 proceeds via an extended nucleus and that the denatured state retains significant malleable structure at the interface between the second and third helices. Our observations constitute the essential prerequisite for structure-based drug-design studies, aimed at identifying molecules that may rescue pathological NPM1 mutants by stabilizing the native-like state

Autotests de diagnostic : une proposition de valeur pour les usagers du système de santé ?

International audienceAt a time when patient empowerment is being talked about as a major trend, diagnostic self-tests might seem like a good option for both the population and healthcare institutions seeking to influence health policies. Such tests enable individuals to assess their own medical condition in an autonomous way, and they could also help control healthcare budgets by providing earlier diagnoses. In France, the market for self-tests has recently expanded with a battery of new tests available for a variety of health conditions. However, these tests are still at an early stage of adoption and have stirred controversy in the medical community with regard to the usability of the information delivered to patients. The present study’s objective is to explore whether the general population between 30 and 50 years of age is ready to adopt these self-tests. A qualitative study using semi-structured interviews was conducted to investigate the thinking of 22 participants living in or around Paris with regard to medical self-testing in general and the latest types of self-tests in particular. The study’s findings highlight the participants’ low awareness of self-tests and their double-edged attitude toward them. The results suggest there is a discrepancy between patients’ stated willingness to emancipate themselves from medical professionals and their level of dependency when it comes to receiving a diagnosis. This finding emphasizes a strong need for guidance by the medical community, as the users do not trust their own skills to analyze self-tests’ results and they are satisfied with traditional laboratory testing

Folding mechanism of the C-terminal domain of nucleophosmin: residual structure in the denatured state and its pathophysiological significance

Nucleophosmin (NPM1) is a ubiquitously expressed protein and is one of the most abundant proteins found in the nucleolus. Naturally occurring mutations in the C-terminal domain of nucleophosmin (Cter-NPM1) are found in similar to 30% of patients with acute myeloid leukemia (AML). These mutations cause changes at the C terminus of NPM1 that lead to denaturation of the protein, a critical factor in determining aberrant translocation of NPM1 to the cytosol. Hence, this protein system represents an ideal candidate to investigate the relations between folding and unfolding and disease. Here we report the characterization of the folding and unfolding kinetics of Cter-NPM1. Data reveal that this small helical domain folds via a compact denatured state, displaying a malleable residual structure. Moreover, analysis of folding rate constants measured under different experimental conditions suggests that the existence of a preorganized structure in the denatured state accelerates folding, implying a native-like residual structure. Because a major feature of Cter-NPM1 mutants responsible for AML is a reduction in stability of the protein and thus prevalence of a denatured state even under physiological conditions, our findings may pave the way to further studies with the aim of designing chemicals capable of interacting with the "pathological" mutants to stabilize the native conformation.-Scaloni, F., Gianni, S., Federici, L., Falini, B., Brunori, M. Folding mechanism of the C-terminal domain of nucleophosmin: residual structure in the denatured state and its pathophysiological significance. FASEB J. 23, 2360-2365 (2009

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns.

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP-PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense

Nucleophosmin C-terminal leukemia-associated domain interacts with G-rich quadruplex forming DNA.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling phosphoprotein, mainly localized at nucleoli, that plays a key role in ribogenesis, centrosome duplication, and response to stress stimuli. Mutations at the C-terminal domain of NPM1 are the most frequent genetic lesion in acute myeloid leukemia and cause the aberrant and stable translocation of the protein in the cytoplasm. The NPM1 C-terminal domain was previously shown to bind nucleic acids. Here we further investigate the DNA binding properties of the NPM1 C-terminal domain both at the protein and nucleic acid levels; we investigate the domain boundaries and identify key residues for high affinity recognition. Furthermore, we demonstrate that the NPM1 C-terminal domain has a preference for G-quadruplex forming DNA regions and induces the formation of G-quadruplex structures in vitro. Finally we show that a specific sequence found at the SOD2 gene promoter, which was previously shown to be a target of NPM1 in vivo, is indeed folded as a G-quadruplex in vitro under physiological conditions. Our data extend considerably present knowledge on the DNA binding properties of NPM1 and suggest a general role in the transcription of genes characterized by the presence of G-quadruplex forming regions at their promoter