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The mal protein, an integral component of specialized membranes, in normal cells and cancer

The MAL gene encodes a 17-kDa protein containing four putative transmembrane segments whose expression is restricted to human T cells, polarized epithelial cells and myelin-forming cells. The MAL protein has two unusual biochemical features. First, it has lipid-like properties that qualify it as a member of the group of proteolipid proteins. Second, it partitions selectively into detergent-insoluble membranes, which are known to be enriched in condensed cell membranes, consistent with MAL being distributed in highly ordered membranes in the cell. Since its original description more than thirty years ago, a large body of evidence has accumulated supporting a role of MAL in specialized membranes in all the cell types in which it is expressed. Here, we review the structure, expression and biochemical characteristics of MAL, and discuss the association of MAL with raft membranes and the function of MAL in polarized epithelial cells, T lymphocytes, and myelin-forming cells. The evidence that MAL is a putative receptor of the epsilon toxin of Clostridium perfringens, the expression of MAL in lymphomas, the hypermethylation of the MAL gene and subsequent loss of MAL expression in carcinomas are also presented. We propose a model of MAL as the organizer of specialized condensed membranes to make them functional, discuss the role of MAL as a tumor suppressor in carcinomas, consider its potential use as a cancer biomarker, and summarize the directions for future researchResearch in the laboratory of MAA was supported by a grant (PGC2018-095643-B-I00) from the Spanish Ministerio de Ciencia e Innovación (MCIN), Agencia Estatal de Investigación, and the Fondo Europeo de Desarrollo Regional, European Union (MCIN/AEI/FEDER, EU). Research in the laboratory of IC was supported by a grant (B2017/BMD-3817) from the Comunidad de Madrid, Spai

Effect of the catalyst system on the reactivity of a polyurethane resin system for RTM manufacturing of structural composites

The high versatility of polyurethanes (PU’s) is encouraging the development of new formulations for new appli cations, like their use as a matrix for structural composites. PU’s based technology offers some advantages, such as fatigue resistance and fast curing cycles. However, their high reactivity hinders some manufacturing processes like Resin Transfer Moulding (RTM). This work aimed to achieve a PU resin (PUR) formulation with the required latency and reactivity for the RTM. For this purpose, different catalytic systems based on an epoxide and LiCl were investigated. The reactivity of the systems was evaluated through Differential Scanning Calorimetry (DSC) and rheology tests, and the curing reaction and viscosity were modelled. Furthermore, the RTM process of a representative composite part was simulated. Results demon strated the processability improvements when the LiCl was incorporated into the isocyanate component of the formulation combined with a monool or a diol. It was observed that these combinations contribute to the encapsulation of the LiCl between the as formed urethane groups by hydrogen bonding, providing the desired latency and acting as a delayed action catalyst. Once the reaction started and the encapsulation was deactivated, an alkoxide was formed to act as a catalyst. En capsulation was more effective with the diol, providing a higher latency.We gratefully acknowledge the Basque Government for the fi nancial support through the ELKARTEK 2020 (ProjectAVAN SITE New generation of sustainable composites for advanced manufacturing KK2020/00019) program. The authors also ac knowledge the University of the Basque Country (UPV/EHU) in the frame of GIU18/216 Research Group and the Macrobe havior-Mesostructure-Nanotechnology SGIker unit

An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

<p>Abstract</p> <p>Background</p> <p>In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.</p> <p>Results</p> <p>By analyzing the <it>in vitro </it>expression of a panel of naturally occurring 4.1R cDNAs, we observed that all AUG1/AUG2-containing cDNAs gave rise to both long, 135 kDa, and short, 80 kDa, 4.1R isoforms. More importantly, similar results were also observed in cells transfected with this set of 4.1R cDNAs. Mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from AUG2. The presence of a cryptic promoter in the 4.1R cDNA sequence was also discounted. When a 583 nt sequence comprised between AUG1 and AUG2 was introduced into bicistronic vectors it directed protein expression from the second cistron. This was also the case when ribosome scanning was abolished by introduction of a stable hairpin at the 5' region of the first cistron. Deletion analysis of the 583 nt sequence indicated that nucleotides 170 to 368 are essential for expression of the second cistron. The polypyrimidine tract-binding protein bound to the 583 nt active sequence but not to an inactive 3'-fragment of 149 nucleotides.</p> <p>Conclusion</p> <p>Our study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of 80 kDa isoforms of protein 4.1R. This mechanism might also account for the generation of 60 kDa isoforms of 4.1R from a downstream AUG3. Our results reveal an additional level of control to 4.1R gene expression pathways and will contribute to the understanding of the biology of proteins 4.1R and their homologues, comprising an ample family of proteins involved in cytoskeletal organization.</p

The MAL family of proteins: Normal function, expression in cancer, and potential use as cancer biomarkers

The MAL family of integral membrane proteins consists of MAL, MAL2, MALL, PLLP, CMTM8, MYADM, and MYADML2. The best characterized members are elements of the machinery that controls specialized pathways of membrane traffic and cell signaling. This review aims to help answer the following questions about the MAL-family genes: (i) is their expression regulated in cancer and, if so, how? (ii) What role do they play in cancer? (iii) Might they have biomedical applications? Analysis of large-scale gene expression datasets indicated altered levels of MAL-family transcripts in specific cancer types. A comprehensive literature search provides evidence of MAL-family gene dysregulation and protein function repurposing in cancer. For MAL, and probably for other genes of the family, dysregulation is primarily a consequence of gene methylation, although copy number alterations also contribute to varying degrees. The scrutiny of the two sources of information, datasets and published studies, reveals potential prognostic applications of MAL-family members as cancer biomarkers—for instance, MAL2 in breast cancer, MAL2 and MALL in pancreatic cancer, and MAL and MYADM in lung cancer—and other biomedical uses. The availability of validated antibodies to some MAL-family proteins sanctions their use as cancer biomarkers in routine clinical practicePID2021-123179NB-I0

An essential role for the MAL protein in targeting Lck to the plasma membrane of human T lymphocytes

The MAL protein is an essential component of the specialized machinery for apical targeting in epithelial cells. The src family kinase Lck plays a pivotal role in T cell signaling. We show that MAL is required in T cells for efficient expression of Lck at the plasma membrane and activation of IL-2 transcription. To investigate the mechanism by which MAL regulates Lck targeting, we analyzed the dynamics of Lck and found that it travels to the plasma membrane in specific transport carriers containing MAL. Coimmunoprecipitation experiments indicated an association of MAL with Lck. Both carrier formation and partitioning of Lck into detergent-insoluble membranes were ablated in the absence of MAL. Polarization of T cell receptor for antigen (TCR) and microtubule-organizing center to immunological synapse (IS) were also defective. Although partial correction of the latter defects was possible by forced expression of Lck at the plasma membrane, their complete correction, formation of transport vesicles, partitioning of Lck, and restoration of signaling pathways, which are required for IL-2 transcription up-regulation, were achieved by exogenous expression of MAL. We concluded that MAL is required for recruitment of Lck to specialized membranes and formation of specific transport carriers for Lck targeting. This novel transport pathway is crucial for TCR-mediated signaling and IS assembly

NMR characterisation of the minimal interacting regions of centrosomal proteins 4.1R and NuMA1: effect of phosphorylation

<p>Abstract</p> <p>Background</p> <p>Some functions of 4.1R in non-erythroid cells are directly related with its distinct sub-cellular localisation during cell cycle phases. During mitosis, 4.1R is implicated in cell cycle progression and spindle pole formation, and co-localizes with NuMA1. However, during interphase 4.1R is located in the nucleus and only partially co-localizes with NuMA1.</p> <p>Results</p> <p>We have characterized by NMR the structural features of the C-terminal domain of 4.1R and those of the minimal region (the last 64 residues) involved in the interaction with NuMA1. This subdomain behaves as an intrinsically unfolded protein containing a central region with helical tendency. The specific residues implicated in the interaction with NuMA1 have been mapped by NMR titrations and involve the N-terminal and central helical regions. The segment of NuMA1 that interacts with 4.1R is phosphorylated during mitosis. Interestingly, NMR data indicates that the phosphorylation of NuMA1 interacting peptide provokes a change in the interaction mechanism. In this case, the recognition occurs through the central helical region as well as through the C-terminal region of the subdomain meanwhile the N-terminal region do not interact.</p> <p>Conclusions</p> <p>These changes in the interaction derived from the phosphorylation state of NuMA1 suggest that phosphorylation can act as subtle mechanism of temporal and spatial regulation of the complex 4.1R-NuMA1 and therefore of the processes where both proteins play a role.</p

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation

The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extensionThis work was supported by the Ministerio de Economıa y Competitividad, Spain [grant numbers BFU2012-32532 and CONSOLIDER COAT CSD2009-00016 to M. A.A.]. G.A. was supported by the Amarouto Program for senior researchers from the Comunidad Autónoma de Madri

MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells

MALL, a membrane-tetra-spanning proteolipid overexpressed in cancer, is present in membraneless nuclear biomolecular condensates

Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning myelin proteolipids, can be converted in vitro into a water-soluble form with a distinct conformation, raising the question of whether these, or other similar proteolipids, can adopt two different conformations in the cell to adapt their structure to distinct environments. Here, we show that MALL, another proteolipid with a membrane-tetra-spanning structure, distributes in membranes outside the nucleus and, within the nucleus, in membrane-less, liquid-like PML body biomolecular condensates. Detection of MALL in one or other environment was strictly dependent on the method of cell fixation used, suggesting that MALL adopts different conformations depending on its physical environment —lipidic or aqueous— in the cell. The acquisition of the condensate-compatible conformation requires PML expression. Excess MALL perturbed the distribution of the inner nuclear membrane proteins emerin and LAP2β, and that of the DNA-binding protein BAF, leading to the formation of aberrant nuclei. This effect, which is consistent with studies identifying overexpressed MALL as an unfavorable prognostic factor in cancer, could contribute to cell malignancy. Our study establishes a link between proteolipids, membranes and biomolecular condensates, with potential biomedical implication