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1.
Acta Neuropathol Commun ; 12(1): 88, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38840253

ABSTRACT

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the coding sequence of huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) of the corpus striatum. No effective treatment is still available, thus urging the identification of potential therapeutic targets. While evidence of mitochondrial structural alterations in HD exists, previous studies mainly employed 2D approaches and were performed outside the strictly native brain context. In this study, we adopted a novel multiscale approach to conduct a comprehensive 3D in situ structural analysis of mitochondrial disturbances in a mouse model of HD. We investigated MSSNs within brain tissue under optimal structural conditions utilizing state-of-the-art 3D imaging technologies, specifically FIB/SEM for the complete imaging of neuronal somas and Electron Tomography for detailed morphological examination, and image processing-based quantitative analysis. Our findings suggest a disruption of the mitochondrial network towards fragmentation in HD. The network of interlaced, slim and long mitochondria observed in healthy conditions transforms into isolated, swollen and short entities, with internal cristae disorganization, cavities and abnormally large matrix granules.


Subject(s)
Disease Models, Animal , Huntington Disease , Imaging, Three-Dimensional , Mitochondria , Animals , Huntington Disease/pathology , Huntington Disease/genetics , Huntington Disease/metabolism , Mitochondria/ultrastructure , Mitochondria/pathology , Mitochondria/metabolism , Imaging, Three-Dimensional/methods , Mice , Mice, Transgenic , Brain/pathology , Brain/ultrastructure , Brain/metabolism , Microscopy, Electron/methods , Male , Neurons/pathology , Neurons/ultrastructure , Neurons/metabolism
2.
Neurobiol Dis ; 195: 106488, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38565397

ABSTRACT

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.


Subject(s)
Disease Models, Animal , Huntington Disease , Polyribosomes , Ribosomes , Animals , Huntington Disease/metabolism , Huntington Disease/pathology , Huntington Disease/genetics , Mice , Polyribosomes/metabolism , Ribosomes/metabolism , Corpus Striatum/metabolism , Corpus Striatum/pathology , Mice, Transgenic , Disease Progression , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Peptide Initiation Factors/metabolism , Peptide Initiation Factors/genetics
3.
Nanomaterials (Basel) ; 11(2)2021 Feb 17.
Article in English | MEDLINE | ID: mdl-33671209

ABSTRACT

Chaperonins are molecular chaperones found in all kingdoms of life, and as such they assist in the folding of other proteins. Structurally, chaperonins are cylinders composed of two back-to-back rings, each of which is an oligomer of ~60-kDa proteins. Chaperonins are found in two main conformations, one in which the cavity is open and ready to recognise and trap unfolded client proteins, and a "closed" form in which folding takes place. The conspicuous properties of this structure (a cylinder containing a cavity that allows confinement) and the potential to control its closure and aperture have inspired a number of nanotechnological applications that will be described in this review.

4.
Bioinformatics ; 36(12): 3947-3948, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32221611

ABSTRACT

SUMMARY: We have developed a software tool to improve the image quality in focused ion beam-scanning electron microscopy (FIB-SEM) stacks: PolishEM. Based on a Gaussian blur model, it automatically estimates and compensates for the blur affecting each individual image. It also includes correction for artifacts commonly arising in FIB-SEM (e.g. curtaining). PolishEM has been optimized for an efficient processing of huge FIB-SEM stacks on standard computers. AVAILABILITY AND IMPLEMENTATION: PolishEM has been developed in C. GPL source code and binaries for Linux, OSX and Windows are available at http://www.cnb.csic.es/%7ejjfernandez/polishem. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Microscopy , Software , Computers , Image Enhancement
5.
F1000Res ; 72018.
Article in English | MEDLINE | ID: mdl-30338057

ABSTRACT

Protein homeostasis (proteostasis) is an essential pillar for correct cellular function. Impairments in proteostasis are encountered both in aging and in several human disease conditions. Molecular chaperones are important players for proteostasis; in particular, heat shock protein 70 (Hsp70) has an essential role in protein folding, disaggregation, and degradation. We have recently proposed a model for Hsp70 functioning as a "multiple socket". In the model, Hsp70 provides a physical platform for the binding of client proteins, other chaperones, and cochaperones. The final fate of the client protein is dictated by the set of Hsp70 interactions that occur in a given cellular context. Obtaining structural information of the different Hsp70-based protein complexes will provide valuable knowledge to understand the functional mechanisms behind the master role of Hsp70 in proteostasis. We additionally evaluate some of the challenges for attaining high-resolution structures of such complexes.


Subject(s)
HSP70 Heat-Shock Proteins/physiology , Proteostasis , Animals , HSP70 Heat-Shock Proteins/chemistry , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/physiology , Multiprotein Complexes/chemistry , Multiprotein Complexes/physiology , Protein Binding , Protein Conformation
6.
FEBS Lett ; 591(17): 2648-2660, 2017 09.
Article in English | MEDLINE | ID: mdl-28696498

ABSTRACT

Proteostasis, the controlled balance of protein synthesis, folding, assembly, trafficking and degradation, is a paramount necessity for cell homeostasis. Impaired proteostasis is a hallmark of ageing and of many human diseases. Molecular chaperones are essential for proteostasis in eukaryotic cells, and their function has traditionally been linked to protein folding, assembly and disaggregation. More recent findings suggest that chaperones also contribute to key steps in protein degradation. In particular, Hsp70 has an essential role in substrate degradation through the ubiquitin-proteasome system, as well as through different autophagy pathways. Accumulated knowledge suggests that the fate of an Hsp70 substrate is dictated by the combination of partners (cochaperones and other chaperones) that interact with Hsp70 in a given cell context.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Proteolysis , Animals , Disease , HSP70 Heat-Shock Proteins/chemistry , Humans , Protein Domains
7.
J Cell Sci ; 130(1): 83-89, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27505890

ABSTRACT

Macroautophagy is morphologically characterized by autophagosome formation. Autophagosomes are double-membraned vesicles that sequester cytoplasmic components for further degradation in the lysosome. Basal autophagy is paramount for intracellular quality control in post-mitotic cells but, surprisingly, the number of autophagosomes in post-mitotic neurons is very low, suggesting that alternative degradative structures could exist in neurons. To explore this possibility, we have examined neuronal subcellular architecture by performing three-dimensional (3D) electron tomography analysis of mouse brain tissue that had been preserved through high-pressure freezing. Here, we report that sequestration of neuronal cytoplasmic contents occurs at the Golgi complex in distinct and dynamic structures that coexist with autophagosomes in the brain. These structures are composed of several concentric double-membraned layers that appear to be formed simultaneously by the direct bending and sealing of discrete Golgi stacks. These structures are labelled for proteolytic enzymes, and lysosomes and late endosomes are found in contact with them, leading to the possibility that the sequestered material could be degraded inside them. Our findings highlight the key role that 3D electron tomography, together with tissue rapid-freezing techniques, will have in gaining new knowledge about subcellular architecture.


Subject(s)
Brain/ultrastructure , Electron Microscope Tomography/methods , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Imaging, Three-Dimensional , Neurons/metabolism , Neurons/ultrastructure , Animals , Cryopreservation , Mice, Inbred C57BL
8.
Protein Eng Des Sel ; 24(1-2): 41-51, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20952436

ABSTRACT

The relevance of p53 as a tumour suppressor is evident from the fact that more than 50% of the human cancers hold mutations in the gene coding for p53, and of the remaining cancers a considerable number have alterations in the p53 pathway. From its discovery 30 years ago, the importance of p53 as an essential transcription factor for tumour suppression has become clear. More recently, new and seemingly diverse roles of p53 have been discovered. It soon became clear that protein-protein interactions play an important role in the regulation of the p53 function at different levels. Here we review the contribution by Prof. Fersht and his group towards understanding the basis and functional relevance of p53 protein-protein interactions, and the important role that protein science, biophysics and structural biology have played in the science produced in the Centre for Protein Engineering over the years.


Subject(s)
Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Humans , Neoplasms/metabolism , Protein Binding , Protein Interaction Mapping , Tumor Suppressor Protein p53/chemistry
9.
Neurobiol Dis ; 41(1): 23-32, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20732420

ABSTRACT

The endoplasmic reticulum-stress response is induced in several neurodegenerative diseases and in cellular models of Huntington's disease. However, here we report that the processing of ATF6α to its active nuclear form, one of the three branches of endoplasmic reticulum-stress activation, is impaired in both animal models and Huntington's disease patients. ATF6α has been reported to be essential for the survival of dormant tumour cells that, like neurons, are arrested in the G0-G1 phase of the cell cycle. This effect is mediated by the small GTPase Rheb (Ras-homologue enriched in brain). Our results suggest that the ATF6α/Rheb pathway is altered in Huntington's disease as the decrease in ATF6α processing is accompanied by a decrease in the accumulation of Rheb. These alterations correlate with the aberrant accumulation of cell cycle re-entry markers in post-mitotic neurons which is accompanied by death of a subset of neurons.


Subject(s)
Activating Transcription Factor 6/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle/genetics , Huntington Disease/metabolism , Monomeric GTP-Binding Proteins/metabolism , Neurons/metabolism , Neuropeptides/metabolism , Activating Transcription Factor 6/genetics , Aged , Aged, 80 and over , Animals , Cell Cycle Proteins/genetics , Female , Humans , Huntington Disease/genetics , Huntington Disease/pathology , Male , Mice , Middle Aged , Monomeric GTP-Binding Proteins/genetics , Neurons/pathology , Neuropeptides/genetics , Ras Homolog Enriched in Brain Protein
10.
Protein Sci ; 17(10): 1663-70, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18694925

ABSTRACT

p53 binds to some members of the S100 family (S100B, S100A4, S100A2, and S100A1). We previously showed that both S100B and S100A4 bind to the p53 tetramerization domain, and consequently control its oligomerization state, but only S100B binds to the C-terminal negative regulatory domain (NRD). Here, we investigate other binding partners for p53 within the S100 family (S100A6 and S100A11), and show that binding to the p53 tetramerization domain seems to be a general feature of the S100 family, while binding to the NRD is a characteristic of a subset of the family.


Subject(s)
S100 Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S100 Calcium-Binding Protein A4 , S100 Proteins/chemistry , Sequence Alignment , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics
11.
Proc Natl Acad Sci U S A ; 102(13): 4735-40, 2005 Mar 29.
Article in English | MEDLINE | ID: mdl-15781852

ABSTRACT

S100B protein is elevated in the brains of patients with early stages of Alzheimer's disease and Down's syndrome. S100A4 is correlated with the development of metastasis. Both proteins bind to p53 tumor suppressor. We found that both S100B and S100A4 bind to the tetramerization domain of p53 (residues 325-355) only when exposed in lower oligomerization states and so they disrupt the tetramerization of p53. In addition, S100B binds to the negative regulatory and nuclear localization domains, which results in a very tight binding to p53 protein sequences that exposed the tetramerization domain in their C terminus. Because the trafficking of p53 depends on its oligomerization state, we suggest that S100B and S100A4 could regulate the subcellular localization of p53. But, the differences in the way these proteins bind to p53 could result in S100B and S1004 having different effects on p53 function in cell-cycle control.


Subject(s)
Polymers/metabolism , S100 Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Fluorescein , Fluorescence Polarization , Gene Components , Humans , Molecular Sequence Data , Nerve Growth Factors , Protein Binding , Protein Structure, Tertiary , S100 Calcium Binding Protein beta Subunit , S100 Calcium-Binding Protein A4 , Tumor Suppressor Protein p53/genetics
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