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Producción Científica, The FASTK family proteins have recently emerged as key post-transcriptional regulators of mitochondrial gene expression. FASTK, the founding member and its homologs FASTKD1–5 are architecturally related RNA-binding proteins, each having a different function in the regulation of mitochondrial RNA biology, from mRNA processing and maturation to ribosome assembly and translation. In this review, we outline the structure, evolution and function of these FASTK proteins and discuss the individual role that each has in mitochondrial RNA biology. In addition, we highlight the aspects of FASTK research that still require more attention., Swiss National Science Foundation (grant 310030B_160257/1), Junta de Castilla y León (grants BIO/VA20/15 and BIO/VA21/15)

Producción Científica, The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria., Junta de Castilla y León (grants BIO/ VA20/15 and BIO/VA21/15), Swiss National Science Foundation (grant 310030B_160257/1)

Hypersensitivity reactions (HSRs) to chemotherapy drugs, such as taxanes and platins, and to monoclonal antibodies limit their therapeutic use due to the severity of some reactions and the fear of inducing a potentially lethal reaction in highly sensitized patients. Patients who experience hypersensitivity reactions face the prospect of abandoning first-line treatment and switching to a second-line, less effective therapy. Some of these reactions are mast cell-mediated hypersensitivity reactions, a subset of which occur through an immunoglobulin (IgE)-dependent mechanism, and are thus true allergies. Others involve mast cells without a demonstrable IgE mechanism. Whether basophils can participate in these reactions has not been demonstrated. Rapid drug desensitization (RDD) is a procedure that induces temporary tolerance to a drug, allowing a medication allergic patient to receive the optimal agent for his or her disease. Through RDD, patients with IgE and non-IgE HSRs can safely be administered important medications while minimizing or completely inhibiting adverse reactions. Due to the clinical expansion and success of RDD, the molecular mechanisms inducing the temporary tolerization have been investigated and are partially understood, allowing for safer and more effective protocols. This article reviews the current literature on molecular mechanisms of RDD with an emphasis in our recent contributions to this field as well as the indications, methods and outcomes of RDD for taxanes, platins, and monoclonal antibodies.

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Gene array analysis revealed that a subset of mRNAs overexpressed in macrophages lacking the destabilizing factor TTP are also overexpressed in macrophages lacking the translational silencer TIA-1. We confirmed that a representative transcript, apobec-1, is significantly stabilized in cells lacking TIA-1. Tethering TIA-1 to a reporter transcript also promotes mRNA decay, suggesting that TIA-1-mediated translational silencing can render mRNA susceptible to the decay machinery. TIA-1-mediated decay is inhibited by small interfering RNAs targeting components of either the 5'-3' (e.g. DCP2) or the 3'-5' (e.g. exosome component Rrp46) decay pathways, suggesting that TIA-1 renders mRNA susceptible to both major decay pathways. TIA-1-mediated decay is inhibited by cycloheximide and emetine, drugs that stabilize polysomes, but is unaffected by puromycin, a drug that disassembles polysomes. These results suggest that TIA-1-induced polysome disassembly is required for enhanced mRNA decay and that TIA-1-induced translational silencing promotes the decay of selected mRNAs.

SAP (SLAM-associated protein) is a small lymphocyte-specific signalling molecule that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). Consistent with its single src homology 2 (SH2) domain architecture and unusually high affinity for SLAM (also called CD150), SAP has been suggested to function by blocking binding of SHP-2 or other SH2-containing signalling proteins to SLAM receptors. Additionally, SAP has recently been shown to be required for recruitment and activation of the Src-family kinase FynT after SLAM ligation. This signalling 'adaptor' function has been difficult to conceptualize, because unlike typical SH2-adaptor proteins, SAP contains only a single SH2 domain and lacks other recognized protein interaction domains or motifs. Here, we show that the SAP SH2 domain binds to the SH3 domain of FynT and directly couples FynT to SLAM. The crystal structure of a ternary SLAM-SAP-Fyn-SH3 complex reveals that SAP binds the FynT SH3 domain through a surface-surface interaction that does not involve canonical SH3 or SH2 binding interactions. The observed mode of binding to the Fyn-SH3 domain is expected to preclude the auto-inhibited conformation of Fyn, thereby promoting activation of the kinase after recruitment. These findings broaden our understanding of the functional repertoire of SH3 and SH2 domains.

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency characterized by extreme susceptibility to Epstein-Barr virus. The XLP disease gene product SH2D1A (SAP) interacts via its SH2 domain with a motif (TIYXXV) present in the cytoplasmic tail of the cell-surface receptors CD150/SLAM, CD84, CD229/Ly-9, and CD244/2B4. Characteristically, the SH2D1A three-pronged interaction with Tyr(281) of CD150 can occur in absence of phosphorylation. Here we analyze the effect of SH2D1A protein missense mutations identified in 10 XLP families. Two sets of mutants were found: (i) mutants with a marked decreased protein half-life (e.g. Y7C, S28R, Q99P, P101L, V102G, and X129R) and (ii) mutants with structural changes that differently affect the interaction with the four receptors. In the second group, mutations that disrupt the interaction between the SH2D1A hydrophobic cleft and Val +3 of its binding motif (e.g. T68I) and mutations that interfere with the SH2D1A phosphotyrosine-binding pocket (e.g. C42W) abrogated SH2D1A binding to all four receptors. Surprisingly, a mutation in SH2D1A able to interfere with Thr -2 of the CD150 binding motif (mutant T53I) severely impaired non-phosphotyrosine interactions while preserving unaffected the binding of SH2D1A to phosphorylated CD150. Mutant T53I, however, did not bind to CD229 and CD224, suggesting that SH2D1A controls several critical signaling pathways in T and natural killer cells. Because no correlation is present between identified types of mutations and XLP patient clinical presentation, additional unidentified genetic or environmental factors must play a strong role in XLP disease manifestations.

The human CD5 lymphocyte cell surface co-receptor modulates activation and differentiation responses mediated by the antigen-specific receptor of T and B cells. CD5 is phosphorylated following lymphocyte activation; however, the exact sites and kinases involved are yet to be determined. Jurkat T cell transfectants expressing tyrosine-mutated CD5 molecules have been used to show that residues Y429 and Y463 are targeted in vivo by protein tyrosine kinases following cell stimulation with anti-CD3 mAb or pervanadate. This is in agreement with data from direct in vitro kinase assays using purified recombinant Lck and Fyn protein tyrosine kinases. The analysis of Lck- and CD3-deficient Jurkat cells shows that tyrosine phosphorylation of CD5 requires Lck activity. We propose that T cell activation mediates CD5 tyrosine phosphorylation at residues Y429 and Y463 mainly through the activation of Lck.

Our understanding of the X-linked lymphoproliferative syndrome (XLP) has advanced significantly in the last two years. The gene that is altered in the condition (SAP/SH2D1A) has been cloned and its protein crystal structure solved. At least two sets of target molecules for this small SH2 domain-containing protein have been identified: A family of hematopoietic cell surface receptors, i.e. the SLAM family, and a second molecule, which is a phosphorylated adapter. A SAP-like protein, EAT-2, has also been found to interact with this family of surface receptors. Several lines of evidence, including structural studies and analyses of missense mutations in XLP patients, support the notion that SAP/SH2D1A is a natural inhibitor of SH2-domain-dependent interactions with members of the SLAM family. However, details of its role in signaling mechanisms are yet to be unravelled. Further analyses of the SAP/SH2D1A gene in XLP patients have made it clear that the development of dys-gammaglobulinemia and B cell lymphoma can occur without evidence of prior EBV infection. Moreover, preliminary results of virus infections of a mouse in which the SAP/SH2D1A gene has been disrupted suggest that EBV infection is not per se critical for the development of XLP phenotypes. It appears therefore that the SAP/SH2D1A gene controls signaling via the SLAM family of surface receptors and thus may play a fundamental role in T cell and APC interactions during viral infections.

Producción Científica, We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST−/−) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST−/− mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST−/− mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-α and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST−/− neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Fas-activated serine/threonine phosphoprotein (FAST) is a survival protein that is tethered to the outer mitochondrial membrane. In cells subjected to environmental stress, FAST moves to stress granules, where it interacts with TIA1 to modulate the process of stress-induced translational silencing. Both FAST and TIA1 are also found in the nucleus, where TIA1 promotes the inclusion of exons flanked by weak splice recognition sites such as exon IIIb of the fibroblast growth factor receptor 2 (FGFR2) mRNA. Two-hybrid interaction screens and biochemical analysis reveal that FAST binds to several alternative and constitutive splicing regulators, suggesting that FAST might participate in this process. The finding that FAST is concentrated at nuclear speckles also supports this contention. We show that FAST, like TIA1, promotes the inclusion of exon IIIb of the FGFR2 mRNA. Both FAST and TIA1 target a U-rich intronic sequence (IAS1) adjacent the 5′ splice site of exon IIIb. However, unlike TIA1, FAST does not bind to the IAS1 sequence. Surprisingly, knockdown experiments reveal that FAST and TIA1 act independently of one another to promote the inclusion of exon IIIb. Mutational analysis reveals that FAST-mediated alternative splicing is separable from the survival effects of FAST. Our data reveal that nuclear FAST can regulate the splicing of FGFR2 transcripts.

The Fas-activated serine/threonine phosphoprotein (FAST) is tethered to the outer mitochondrial membrane, where it interacts with BCL-X(L) (17). Here we show that RNA interference-mediated knockdown of endogenous FAST results in apoptosis, whereas overexpressed recombinant FAST inhibits Fas- and UV-induced apoptosis, indicating that FAST is a survival protein. The antiapoptotic effects of FAST are regulated by interactions with the translational silencer TIA-1: a FAST mutant lacking its TIA-1-binding domain does not inhibit apoptosis, and overexpressed recombinant TIA-1 inhibits the antiapoptotic effects of FAST. Because the antiapoptotic effects of FAST require ongoing protein synthesis, we hypothesized that FAST might function by preventing TIA-1-mediated silencing of mRNAs encoding inhibitors of apoptosis. Consistent with this hypothesis, FAST promotes the expression of cotransfected reporter proteins, a process that requires its TIA-1-binding domain and is inhibited by overexpressed recombinant TIA-1. More compellingly, recombinant FAST increases the expression of endogenous cIAP-1 and XIAP, but not GAPDH, in transfected HeLa cells. Because FAST is released from mitochondria in cells undergoing Fas- or UV-induced apoptosis, we propose that FAST serves as a sensor of mitochondrial stress that modulates a TIA-1-regulated posttranscriptional stress response program.

CD150 signaling lymphocytic activation molecule (SLAM), a T/B/dendritic cell surface glycoprotein, is a costimulatory receptor involved in T-cell activation and is also a receptor for measles virus. CD150-induced signal transduction is controlled bySAP/SH2D1A, the gene that is aberrant in X-linked lymphoproliferative disease and familial hemophagocytic lymphohistiocytosis. This report shows that CD150 colocalizes with the T-cell receptor (TCR) following CD3 triggering in human peripheral blood T cells and is rapidly and reversibly tyrosine phosphorylated on TCR cross-linking. The Src-like kinases Lck and Fyn phosphorylate tyrosine residues in the cytoplasmic tail of CD150. The results demonstrate that the SAP protein has 2 modes of binding to CD150. Binding to the motif Thr-Ile-Tyr281Ala-Gln-Val occurs in a phosphotyrosine-independent fashion and to the motif Thr-Val-Tyr327Ala-Ser-Val in a phosphotyrosine-dependent manner. Within both SAP binding motifs the threonine residue at position −2 to tyrosine is essential to stabilize the interaction irrespective of tyrosine phosphorylation, a feature unique to the SAP SH2 domain. A leucine residue, Leu278, further stabilizes nonphospho binding of SAP to Tyr281 of CD150. SAP blocking of the tyrosine phosphatase SHP-2 occurs primarily on Tyr281 of CD150 because SHP-2 requires both Tyr281 and Tyr327 for binding to CD150, and SAP binds to nonphosphorylated Tyr281. CD150 exhibits lateral mobility, segregating into intercellular contacts. The lateral mobility and homophilic clustering of CD150 between neighboring cells is not dependent on SAP/CD150 interaction.

X-linked lymphoproliferative disease (XLP) is a rare immune disorder commonly triggered by infection with Epstein-Barr virus. Major disease manifestations include fatal acute infectious mononucleosis, B-cell lymphoma, and progressive dys-gammaglobulinemia. SAP/SH2D1A, the product of the gene mutated in XLP, is a small protein that comprises a single SH2 domain and a short tail of 26 amino acids. SAP binds to a specific motif in the cytoplasmic tails of the cell surface receptors SLAM and 2B4, where it blocks recruitment of the phosphatase SHP-2. Here it is reported that Ly-9 and CD84, 2 related glycoproteins differentially expressed on hematopoietic cells, also recruit SAP. Interactions between SAP and Ly-9 or CD84 were analyzed using a novel yeast 2-hybrid system, by COS cell transfections and in lymphoid cells. Recruitment of SAP is most efficient when the specific tyrosine residues in the cytoplasmic tails of Ly-9 or CD84 are phosphorylated. It is concluded that in activated T cells, the SAP protein binds to and regulates signal transduction events initiated through the engagement of SLAM, 2B4, CD84, and Ly-9. This suggests that combinations of dysfunctional signaling pathways initiated by these 4 cell surface receptors may cause the complex phenotypes of XLP. (Blood. 2001;97:3867-3874)