Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins

The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that are often responsible for the biological function of oligomeric membrane protein states

Towards Understanding the Molecular Bases of Stretch Activation: A Structural comparison of the Two Troponin C Isoforms of Lethocerus.

Muscles are usually activated by calcium binding to the calcium sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle activation is also produced by mechanical stress. Little is known about the molecular bases of this calcium-independent activation. In Lethocerus, a giant water bug often used as a model system because of its large muscle fibers, there are two troponin-C isoforms, called F1 and F2, that have distinct roles in activating the muscle. It has been suggested that this can be explained either by differences in structural features or by differences in the interactions with other proteins. Here we have compared the structural and dynamic properties of the two proteins and shown how they differ. We have also mapped the interactions of the F2 isoform with peptides spanning the sequence of its natural partner, troponin-I. Our data have allowed us to build a model of the troponin complex and may eventually help in understanding the specialized function of the F1 and F2 isoforms and the molecular mechanism of stretch activation

Genome-wide expression profiling of B lymphocytes reveals IL4R increase in allergic asthma

[EN] Allergic asthma is a multifactorial disorder in which activation and differentiation of B lymphocytes toward the production of IgE play an essential role. In these B cells, molecular mechanisms to generate IgE antibodies from IgM depend on a complex network of molecules that trigger class-switch recombination specifically at the Sε region of the IgH locus. Several trans-membrane receptors and activating signaling cascades are known to physiologically participate in these processes, including the IL4R signaling pathway. In this regard, our group has studied in the past genetic polymorphisms in the IL4/IL4R pathway that might hold clinical correlations with atopy and asthma.Spanish Ministry of Health (FIS - Fondo de Investigación Sanitaria); Instituto de Salud Carlos III; FEDER - Fondo Europeo de Desarrollo Regional funds; Spanish Society of Allergology and Clinical Immunolog

Spatiotemporal progression of ubiquitin-proteasome system inhibition after status epilepticus suggests protective adaptation against hippocampal injury.

BACKGROUND: The ubiquitin-proteasome-system (UPS) is the major intracellular pathway leading to the degradation of unwanted and/or misfolded soluble proteins. This includes proteins regulating cellular survival, synaptic plasticity and neurotransmitter signaling; processes controlling excitability thresholds that are altered by epileptogenic insults. Dysfunction of the UPS has been reported to occur in a brain region- and cell-specific manner and contribute to disease progression in acute and chronic brain diseases. Prolonged seizures, status epilepticus, may alter UPS function but there has been no systematic attempt to map when and where this occurs in vivo or to determine the consequences of proteasome inhibition on seizure-induced brain injury. METHOD: To determine whether seizures lead to an impairment of the UPS, we used a mouse model of status epilepticus whereby seizures are triggered by an intra-amygdala injection of kainic acid. Status epilepticus in this model causes cell death in selected brain areas, in particular the ipsilateral CA3 subfield of the hippocampus, and the development of epilepsy after a short latent period. To monitor seizure-induced dysfunction of the UPS we used a UPS inhibition reporter mouse expressing the ubiquitin fusion degradation substrate ubiquitin(G76V)-green fluorescent protein. Treatment with the specific proteasome inhibitor epoxomicin was used to establish the impact of proteasome inhibition on seizure-induced pathology. RESULTS AND CONCLUSIONS: Our studies show that status epilepticus induced by intra-amygdala kainic acid causes select spatio-temporal UPS inhibition which is most evident in damage-resistant regions of the hippocampus, including CA1 pyramidal and dentate granule neurons then appears later in astrocytes. In support of this exerting a beneficial effect, injection of mice with the proteasome inhibitor epoxomicin protected the normally vulnerable hippocampal CA3 subfield from seizure-induced neuronal death in the model. These studies reveal brain region- and cell-specific UPS impairment occurs after seizures and suggest UPS inhibition can protect against seizure-induced brain damage. Identifying networks or pathways regulated through the proteasome after seizures may yield novel target genes for the treatment of seizure-induced cell death and possibly epilepsy

Additional chromosome abnormalities in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy

Background Acute promyelocytic leukemia is a subtype of acute myeloid leukemia characterized by the t(15;17). The incidence and prognostic significance of additional chromosomal abnormalities in acute promyelocytic leukemia is still a controversial matter. Design and Methods Based on cytogenetic data available for 495 patients with acute promyelocytic leukemia enrolled in two consecutive PETHEMA trials (LPA96 and LPA99), we analyzed the incidence, characteristics, and outcome of patients with acute promyelocytic leukemia with and without additional chromosomal abnormalities who had been treated with all-trans retinoic acid plus anthracycline monochemotherapy for induction and consolidation. Results Additional chromosomal abnormalities were observed in 140 patients (28%). Trisomy 8 was the most frequent abnormality (36%), followed by abn(7q) (5%). Patients with additional chromosomal abnormalities more frequently had coagulopathy (P=0.03), lower platelet counts (P=0.02), and higher relapse-risk scores (P=0.02) than their counterparts without additional abnormalities. No significant association with FLT3/ITD or other clinicopathological characteristics was demonstrated. Patients with and without additional chromosomal abnormalities had similar complete remission rates (90% and 91%, respectively). Univariate analysis showed that additional chromosomal abnormalities were associated with a lower relapse-free survival in the LPA99 trial (P=0.04), but not in the LPA96 trial. However, neither additional chromosomal abnormalities overall nor any specific abnormality was identified as an independent risk factor for relapse in multivariate analysis. Conclusions The lack of independent prognostic value of additional chromosomal abnormalities in acute promyelocytic leukemia does not support the use of alternative therapeutic strategies when such abnormalities are found

WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy. Amoeboid cells are associated with melanoma invasive capacity. Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours

Prognostic utility of serum free light chain ratios and heavy-light chain ratios in multiple myeloma in three PETHEMA/GEM phase III clinical trials

We investigated the prognostic impact and clinical utility of serum free light chains (sFLC) and serum heavy-light chains (sHLC) in patients with multiple myeloma treated according to the GEM2005MENOS65, GEM2005MAS65, and GEM2010MAS65 PETHEMA/GEM phase III clinical trials. Serum samples collected at diagnosis were retrospectively analyzed for sFLC (n = 623) and sHLC (n = 183). After induction or autologous transplantation, 309 and 89 samples respectively were available for sFLC and sHLC assays. At diagnosis, a highly abnormal (HA) sFLC ratio (sFLCr) (32) was not associated with higher risk of progression. After therapy, persistence of involved-sFLC levels >100 mg/L implied worse survival (overall survival [OS], P = 0.03; progression-free survival [PFS], P = 0.007). Among patients that achieved a complete response, sFLCr normalization did not necessarily indicate a higher quality response. We conducted sHLC investigations for IgG and IgA MM. Absolute sHLC values were correlated with monoclonal protein levels measured with serum protein electrophoresis. At diagnosis, HA-sHLCrs (73) showed a higher risk of progression (P = 0.006). Additionally, involved-sHLC levels >5 g/L after treatment were associated with shorter survival (OS, P = 0.001; PFS, P = 0.018). The HA-sHLCr could have prognostic value at diagnosis; absolute values of involved-sFLC >100 mg/L and involved-sHLC >5 g/L could have prognostic value after treatment

Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma

Detecting persistent minimal residual disease (MRD) allows the identification of patients with an increased risk of relapse and death. In this study, we have evaluated MRD 3 months after transplantation in 106 myeloma patients using a commercial next-generation sequencing (NGS) strategy (LymphoTrack®), and compared the results with next-generation flow (NGF, EuroFlow). The use of different marrow pulls and the need of concentrating samples for NGS biased the applicability for MRD evaluation and favored NGF. Despite that, correlation between NGS and NGF was high (R = 0.905). The 3-year progression-free survival (PFS) rates by NGS and NGF were longer for undetectable vs. positive patients (NGS: 88.7% vs. 56.6%; NGF: 91.4% vs. 50%; p < 0.001 for both comparisons), which resulted in a 3-year overall survival (OS) advantage (NGS: 96.2% vs. 77.3%; NGF: 96.6% vs. 74.9%, p < 0.01 for both comparisons). In the Cox regression model, NGS and NGF negativity had similar results but favoring the latter in PFS (HR: 0.20, 95% CI: 0.09-0.45, p < 0.001) and OS (HR: 0.21, 95% CI: 0.06-0.75, p = 0.02). All these results reinforce the role of MRD detection by different strategies in patient prognosis and highlight the use of MRD as an endpoint for multiple myeloma treatment

Spanish guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies