Calreticulin Enhances the Transcriptional Activity of Thyroid Transcription Factor-1 by Binding to Its Homeodomain

Transcription factors are often regulated by associated protein cofactors that are able to modify their activity by several different mechanisms. In this study we show that calreticulin, a Ca2+-binding protein with chaperone activity, binds to thyroid transcription factor-1 (TTF-1), a homeodomain-containing protein implicated in the differentiation of lung and thyroid. The interaction between calreticulin and TTF-1 appears to have functional significance because it results in increased transcriptional stimulation of TTF-1-dependent promoters. Calreticulin binds to the TTF-1 homeodomain and promotes its folding, suggesting that the mechanism involved in stimulation of transcriptional activity is an increase of the steady-state concentration of active TTF-1 protein in the cell. We also demonstrate that calreticulin mRNA levels in thyroid cells are under strict control by the thyroid-stimulating hormone, thus implicating calreticulin in the modulation of thyroid gene expression by thyroid-stimulating hormone

Role of phase partitioning in coordinating DNA damage response: Focus on the Apurinic Apyrimidinic Endonuclease 1 interactome

4noLiquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions while excluding noninteracting components. Disordered domains of proteins, as well as interaction with RNA, favor condensation but are not mandatory for modulating this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses, conferring both spatial and temporal fine regulation. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway, bearing additional 'non-canonical' DNA-repair functions associated with processes like RNA metabolism. Recently, it has been highlighted that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in different pathogenic events. This work might represent a paradigmatical pipeline for evaluating the relevance of LLPS in DDR.openopenTosolini D.; Antoniali G.; Dalla E.; Tell G.Tosolini, D.; Antoniali, G.; Dalla, E.; Tell, G

The Intracellular Localization of APE 1 /Ref-1: More than a Passive Phenomenon?

Human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a perfect paradigm of the functional complexity of a biological macromolecule. First, it plays a crucial role, by both redox-dependent and –independent mechanisms, as a transcriptional coactivator for different transcription factors, either ubiquitous (i.e., AP-1, Egr-1, NF-κB, p53, HIF) or tissue-specific (i.e., PEBP-2, Pax-5 and -8, TTF-1), in controlling different cellular processes such as apoptosis, proliferation, and differentiation. Second, it acts, as an apurinic/apyrimidinic endonuclease, during the second step of the DNA base excision repair pathway, which is responsible for the repair of cellular alkylation and oxidative DNA damages. Third, it controls the intracellular reactive oxygen species production by negatively regulating the activity of the Ras-related GTPase Rac1. Despite these known functions of APE1/Ref-1, information is still scanty about the molecular mechanisms responsible for the coordinated control of its several activities. Some evidence suggests that the expression and subcellular localization of APE1/Ref-1 are finely tuned. APE1/Ref-1 is a ubiquitous protein, but its expression pattern differs according to the different cell types. APE1/Ref-1 subcellular localization is mainly nuclear, but cytoplasmic staining has also been reported, the latter being associated with mitochondria and/or presence within the endoplasmic reticulum. It is not by chance that both expression and subcellular localization are altered in several metabolic and proliferative disorders, such as in tumors and aging. Moreover, a fundamental role played by different posttranslational modifications in modulating APE1/Ref-1 functional activity is becoming evident. In the present review, we tried to put together a growing body of information concerning APE1/Ref-1's different functions, shedding new light on present and future directions to understand fully this unique molecule

Mitochondrial translocation of APE1 relies on the MIA pathway

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1's mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival

the redox sensitive ape1 is a master cellular regulator for inflammatory pain condition

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APE1 polymorphic variants cause persistent genomic stress and affect cancer cell proliferation

Apurinic/apyrimidinic endonuclease 1 (APE1) is the main mammalian AP-endonuclease responsible for the repair of endogenous DNA damage through the base excision repair (BER) pathway. Molecular epidemiological studies have identified several genetic variants associated with human diseases, but a well-defined functional connection between mutations in APE1 and disease development is lacking. In order to understand the biological consequences of APE1 genetic mutations, we examined the molecular and cellular consequences of the selective expression of four non-synonymous APE1 variants (L104R, R237C, D148E and D283G) in human cells. We found that D283G, L104R and R237C variants have reduced endonuclease activity and impaired ability to associate with XRCC1 and DNA polymerase \u3b2, which are enzymes acting downstream of APE1 in the BER pathway. Complementation experiments performed in cells, where endogenous APE1 had been silenced by shRNA, showed that the expression of these variants resulted in increased phosphorylation of histone H2Ax and augmented levels of poly(ADP-ribosyl)ated (PAR) proteins. Persistent activation of DNA damage response markers was accompanied by growth defects likely due to combined apoptotic and autophagic processes. These phenotypes were observed in the absence of exogenous stressors, suggesting that chronic replication stress elicited by the BER defect may lead to a chronic activation of the DNA damage response. Hence, our data reinforce the concept that non-synonymous APE1 variants present in the human population may act as cancer susceptibility alleles

High Mobility Group I Proteins Interfere with the Homeodomains Binding to DNA

Homeodomains (HDs) constitute the DNA binding domain of several transcription factors that control cell differentiation and development in a wide variety of organisms. Most HDs recognize sequences that contain a 5'-TAAT-3' core motif. However, the DNA binding specificity of HD-containing proteins does not solely determine their biological effects, and other molecular mechanisms should be responsible for their ultimate functional activity. Interference by other factors in the HD/DNA interaction could be one of the processes by which HD-containing proteins achieve the functional complexity required for their effects on the expression of target genes. Using gel-retardation assay, we demonstrate that two members of the high mobility group I (HMGI) family of nuclear proteins (HMGI-C and HMGY) can bind to a subset of HD target sequences and inhibit HDs from binding to the same sequences. The inhibition of the HD/DNA interaction occurs while incubating HMGI-C with DNA either before or after the addition of the HD. The reduced half-life of the HD.DNA complex in the presence of HMGI-C, and the shift observed in the CD spectra recorded upon HMGI-C binding to DNA, strongly suggest that structural modifications of the DNA are responsible for the inhibition of the HD.DNA complex formation. Moreover, by co-transfection experiments we provide evidence that this inhibition can occur also in vivo. The data reported here would suggest that HMGI proteins may be potential regulators of the function of HD-containing proteins and that they are able to interfere with the access of the HD to their target genes

Specific Inhibition of the Redox Activity of Ape1/Ref-1 by E3330 Blocks Tnf-A-Induced Activation of Il-8 Production in Liver Cancer Cell Lines

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases

Platinum Salts in Patients with Breast Cancer: A Focus on Predictive Factors

Breast cancer (BC) is the most frequent oncologic cause of death among women and the improvement of its treatments is compelling. Platinum salts (e.g., carboplatin, cisplatin, and oxaliplatin) are old drugs still used to treat BC, especially the triple-negative subgroup. However, only a subset of patients see a concrete benefit from these drugs, raising the question of how to select them properly. Therefore, predictive biomarkers for platinum salts in BC still represent an unmet clinical need. Here, we review clinical and preclinical works in order to summarize the current evidence about predictive or putative platinum salt biomarkers in BC. The association between BRCA1/2 gene mutations and platinum sensitivity has been largely described. However, beyond the mutations of these two genes, several other proteins belonging to the homologous recombination pathways have been linked to platinum response, defining the concept of BRCAness. Several works, here reviewed, have tried to capture BRCAness through different strategies, such as homologous recombination deficiency (HRD) score and genetic signatures. Moreover, p53 and its family members (p63 and p73) might also be used as predictors of platinum response. Finally, we describe the mounting preclinical evidence regarding base excision repair deficiency as a possible new platinum biomarker