Lafora disease (LD), a fatal neurodegenerative disorder characterized by the presence of intracellular inclusions called Lafora bodies (LBs), is caused by loss-of-function mutations in laforin or malin. Previous studies suggested a role of these proteins in the regulation of glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of the intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. We have generated a malin-deficient (Epm2b-/-) mouse with a phenotype similar to that of LD patients. By 3-6 months of age, Epm2b-/- mice present neurological and behavioral abnormalities that correlate with a massive presence of LBs in the cortex, hippocampus and cerebellum. Sixteen-day-old Epm2b-/- mice, without detectable LBs, show an impairment of macroautophagy (hereafter called autophagy), which remains compromised in adult animals. These data demonstrate similarities between the Epm2a-/- and Epm2b-/- mice that provide further insights into LD pathogenesis. They illustrate that the dysfunction of autophagy is a consequence of the lack of laforin-malin complexes and a common feature of both mouse models of LD. Because this dysfunction precedes other pathological manifestations, we propose that decreased autophagy plays a primary role in the formation of LBs and it is critical in LD pathogenesis.
Cyclopentenone prostanoids (cyP) arise as important modulators of inflammation and cell proliferation. Although their physiological significance has not been fully elucidated, their potent biological effects have spurred their study as leads for the development of therapeutic agents. A key determinant of cyP action is their ability to bind to thiol groups in proteins or in glutathione through Michael addition. Even though several protein targets for cyP addition have been identified, little is known about the structural determinants from the protein or the cyP that drive this modification. The results herein presented provide the first evidence that cyP with different structures target distinct thiol sites in a protein molecule, namely, H-Ras. Whereas 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and Delta12-PGJ2 preferentially target the C-terminal region containing cysteines 181 and 184, PGA1 and 8-iso-PGA1 bind mainly to cysteine 118, located in the GTP-binding motif. The biological counterparts of this specificity are the site-selective modification and activation of H-Ras in cells and the differential interaction of cyP with H, N, and K-Ras proteins. Cysteine 184 is unique to H-Ras, whereas cysteine 118 is present in the three Ras homologues. Consistent with this, PGA1 binds to and activates H-, N-, and K-Ras, thus differing from the preferential interaction of 15d-PGJ2 with H-Ras. These results put forward the possibility of influencing the selectivity of cyP-protein addition by modifying cyP structure. Furthermore, they may open new avenues for the development of cyP-based drugs.
Triple-negative breast cancer (TNBC) requires the iden- tification of reliable predictors of response to neoadjuvant chemotherapy (NACT). For this purpose, we aimed to evaluate the performance of the TNBCtype-4 classifier in a cohort of patients with TNBC treated with neoadjuvant carboplatin and docetaxel (TCb). Patients with TNBC were accrued in a nonrandomized trial of neoadjuvant carboplatin AUC 6 and docetaxel 75 mg/m for six cycles. Response was evaluated in terms of pathologic complete response (pCR, ypT0/is ypN0) and residual cancer burden by Symmans and colleagues. Lehmann's subtyping was performed using the TNBCtype online tool from RNAseq data, and germline sequencing of a panel of seven DNA damage repair genes was conducted. Ninety-four out of the 121 patients enrolled in the trial had RNAseq available. The overall pCR rate was 44.7%. Lehmann subtype distribution was 34.0% BL1, 20.2% BL2, 23.4% M, 14.9% LAR, and 7.4% were classified as ER+. Response to NACT with TCb was significantly associated with Lehmann subtype ( = 0.027), even in multivariate analysis including tumor size and nodal involvement, with BL1 patients achieving the highest pCR rate (65.6%), followed by BL2 (47.4%), M (36.4%), and LAR (21.4%). BL1 was associated with a significant younger age at diagnosis and higher ki67 values. Among our 10 germline mutation carriers, 30% were BL1, 40% were BL2, and 30% were M. TNBCtype-4 is associated with significantly different pCR rates for the different subtypes, with BL1 and LAR displaying the best and worse responses to NACT, respectively. .
Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.
Lafora progressive myoclonus epilepsy (Lafora disease) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. The vast majority of patients carry mutations in either the EPM2A or EPM2B genes, encoding laforin, a glucan phosphatase, and malin, an E3 ubiquitin ligase, respectively. Although the precise physiological role of these proteins is not fully understood, work in past years has established a link between glycogen synthesis, Lafora bodies formation and Lafora disease development. To determine the role of the phosphatase activity of laforin in disease development we generated two Epm2a(-/-) mouse lines expressing either wild-type laforin or a mutant (C265S) laforin lacking only the phosphatase activity. Our results demonstrate that expression of either transgene blocks formation of Lafora bodies and restores the impairment in macroautophagy, preventing the development of Lafora bodies in Epm2a(-/-) mice. These data indicate that the critical pathogenic process is the control of abnormal glycogen accumulation through intracellular proteolytic systems by the laforin-malin complex, and not glycogen dephosphorylation by laforin. Understanding which is the essential process leading to Lafora disease pathogenesis represents a critical conceptual advance that should facilitate development of appropriate therapeutics.
Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-D 12,14 -PGJ 2 (15d-PGJ 2 -B) and PGA 1 (PGA 1 -B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ 2 -B and PGA 1 -B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.
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