Antonio Cuadrado

Bio: Antonio Cuadrado is an academic researcher from Spanish National Research Council. The author has contributed to research in topics: Oxidative stress & Transcription factor. The author has an hindex of 62, co-authored 158 publications receiving 12673 citations. Previous affiliations of Antonio Cuadrado include Autonomous University of Madrid & Hospital Universitario La Paz.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases

Abstract: The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), are critical in the maintenance of redox, metabolic and protein homeostasis, as well as the regulation of inflammation. Thus, NRF2 activation provides cytoprotection against numerous pathologies including chronic diseases of the lung and liver; autoimmune, neurodegenerative and metabolic disorders; and cancer initiation. One NRF2 activator has received clinical approval and several electrophilic modifiers of the cysteine-based sensor KEAP1 and inhibitors of its interaction with NRF2 are now in clinical development. However, challenges regarding target specificity, pharmacodynamic properties, efficacy and safety remain.

SCF/{beta}-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner.

Abstract: A disadvantage of aerobic life is the constant generation of potentially damaging reactive oxygen species (ROS). The intracellular levels of such species need to be tightly controlled in order to avoid oxidative stress. Transcription factor Nrf2 (NF-E2-related factor 2) plays a critical role in redox homeostasis since it increases the expression of many antioxidant and drug-metabolizing genes, including those encoding heme oxygenase 1 (HO-1), NADPH:quinone oxidoreductase 1, glutathione S-transferases, glutamate-cysteine ligase, and glutathione peroxidases, in response to oxidative and electrophile stressors (13). These genes all contain a common promoter enhancer called the antioxidant response element (ARE) and are transactivated by Nrf2. Because ROS play a role as intracellular signaling molecules for many physiological processes, Nrf2 can have an impact on numerous cell functions, ranging from differentiation and development to proliferation and inflammation. Therefore, Nrf2 activity influences neurodegenerative disease, cardiovascular disease, and cancer (3, 4, 14, 16, 17, 49, 53). While increased Nrf2 transcriptional activity enhances cellular antioxidant defenses and increases the capacity to detoxify drugs, it may also lead to unwanted side effects. For instance, in tumors, high levels of Nrf2 activity have been correlated with a poor prognosis (41). Indeed, high Nrf2 activity has not been favored during evolution (25), but its levels are restricted via both redox-dependent and redox-independent pathways in normal healthy cells (29). In normal cells, Keap1 (Kelch-like ECH-associated protein 1), an E3 ubiquitin ligase substrate adaptor, regulates the level of Nrf2 protein in a redox-dependent fashion (5, 20, 51). The interaction between Nrf2 and Keap1 occurs via a “two-site tethering” process, otherwise called the “hinge and latch” mechanism. In this model, two motifs, a high-affinity ETGE motif and a low-affinity DLG motif, within the N-terminal Neh2 domain of Nrf2 each interact with a separate Kelch repeat domain present in the Keap1 homodimer (40). Both the ETGE motif and the DLG motif are required for the transcription factor to be repressed by Keap1 (28). In addition to its interaction with Nrf2, Keap1 also binds Cullin 3 (Cul3), which forms a core E3 ubiquitin ligase complex through an association with Ring-box1 protein (Rbx1, also called Roc1) (5, 10, 20, 51). The Keap1-Cul3-Rbx1 complex is able to ubiquitinate Nrf2 and target it for proteasomal degradation only under normal redox conditions, and upon exposure to oxidants or electrophiles, Cys-151, Cys-273, and Cys-288 in Keap1 become modified, leading to disturbance of the interaction between Nrf2 and Keap1 (8, 21, 49, 50). Failure of Nrf2 to dock simultaneously onto both Kelch repeat domains enables it to escape ubiquitination by Cul3-Rbx1 (21, 32, 47, 50). Thus, stress-related modification of Keap1 results in Nrf2 stabilization, accumulation of the transcription factor in the nucleus, and upregulation of ARE-driven genes. Perturbation of the Nrf2-Keap1 complex by oxidants and electrophiles is considered the principal mechanism by which Nrf2 accumulates and induces the ARE-gene battery. However, other regulatory mechanisms must exist in order to explain the following: (i) how Nrf2 contributes to the basal expression of certain ARE-driven genes under normal homeostatic conditions, (ii) how Nrf2 activity returns to its low basal levels after the intracellular redox balance has been restored, and (iii) how Nrf2 activity is limited during oxidative and electrophile stress. Conventional cell signaling studies have suggested that Nrf2 might be regulated by protein phosphorylation (2, 6, 15, 18, 36, 44). Previously, we presented data suggesting that GSK-3β (glycogen synthase kinase 3β) influences the nuclear exclusion and inactivation of Nrf2 (37-39). However, the mechanistic connection between GSK-3 and Nrf2 remains largely unexplored. A number of studies have demonstrated that GSK-3 directs the ubiquitination and proteasomal degradation of various transcription factors and other proteins by SCF/β-TrCP; these include Snail (54), β-catenin (1, 22, 34), Gli2 and Gli3 (33, 48), Xom (55), Cdc 25a (19), FGD1 and -3 (11, 12), Mcl-1 (7), securin (24), prolactin receptor (46), and the phosphatase PHLPP1 (23). In these instances, GSK-3 phosphorylates a cluster of Ser/Thr residues in target proteins, which are then recognized by SCF/β-TrCP. In turn, the complex formed by SCF/β-TrCP binds the Cullin-1 (Cul1) scaffold protein to form a complete E3 ligase by association with a linker protein called Skp1 and with Rbx1. Therefore, β-TrCP is an adapter protein that contains a Skp1-binding site called F-box and a WD recognition domain for phosphorylated substrates in the consensus motif DpSGX(1-4)pS (9, 42). To date, the existence of a phosphodegron in Nrf2 has not been explored. In the present article, we report that Nrf2 is destabilized as a consequence of its phosphorylation by GSK-3 and subsequent ubiquitination by SCF/β-TrCP. This pathway represents an alternative mechanism to the Keap1-dependent degradation of Nrf2 and provides a means by which this transcription factor can be regulated in a redox-independent manner.

Clinical Relevance of Biomarkers of Oxidative Stress

Abstract: Significance: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. Recent Advances: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. Critical Issues: The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clin...

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity

Abstract: The clinical use of anthracyclines like doxorubicin and daunorubicin can be viewed as a sort of double-edged sword. On the one hand, anthracyclines play an undisputed key role in the treatment of many neoplastic diseases; on the other hand, chronic administration of anthracyclines induces cardiomyopathy and congestive heart failure usually refractory to common medications. Second-generation analogs like epirubicin or idarubicin exhibit improvements in their therapeutic index, but the risk of inducing cardiomyopathy is not abated. It is because of their janus behavior (activity in tumors vis-a-vis toxicity in cardiomyocytes) that anthracyclines continue to attract the interest of preclinical and clinical investigations despite their longer-than-40-year record of longevity. Here we review recent progresses that may serve as a framework for reappraising the activity and toxicity of anthracyclines on basic and clinical pharmacology grounds. We review 1) new aspects of anthracycline-induced DNA damage in cancer cells; 2) the role of iron and free radicals as causative factors of apoptosis or other forms of cardiac damage; 3) molecular mechanisms of cardiotoxic synergism between anthracyclines and other anticancer agents; 4) the pharmacologic rationale and clinical recommendations for using cardioprotectants while not interfering with tumor response; 5) the development of tumor-targeted anthracycline formulations; and 6) the designing of third-generation analogs and their assessment in preclinical or clinical settings. An overview of these issues confirms that anthracyclines remain "evergreen" drugs with broad clinical indications but have still an improvable therapeutic index.