Quick two-dimensional differential in gel electrophoresis-based method to determine length and secondary structures of telomeric DNA

A novel differential in gel electrophoresis (DIGE)-based method has been developed and applied to measure telomere length and appearance of two-dimensional structural DNA changes. It can be applied to any area requiring quick and evident measurement of structural DNA changes.     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.Deregulation of the epidermal growth factor receptor signaling network contributes to initiate and/or maintain malignant growth (1). One of these alterations, aberrant cellular motility, is necessary for invasive growth, which eventually culminates with the establishment of distant metastases, the leading cause of death in patients with cancer.The epidermal growth factor receptor is the prototype of a family that also includes HER2 (ErbB2, Neu), HER3, and HER4 (ErbB3 and ErbB4). The analysis of cells expressing various HER receptors indicated that HER2 plays a critical role in the regulation of motility (2, 3). Upon activation through homo- or heterodimerization with other HER receptors, several tyrosines in the cytoplasmic tail of HER2 are phosphorylated and initiate intracellular signaling pathways, including the phospholipase C-γ1 and phosphatidylinositol 3-kinase pathways (4), which, in turn, promote cell migration through partially understood cascades. These cascades are largely regulated by phosphorylation/dephosphorylation of cellular components.A subgroup of HER2-positive patients expresses a series of carboxyl-terminal fragments (CTFs)⁵ of HER2. HER2 CTFs can be generated by two independent mechanisms: proteolytic processing and alternative initiation of translation. Metalloproteases with the so-called α-secretase activity shed the extracellular domain of HER2, leaving behind a fragment, known as P95, that starts around alanine 648 (5) (see also Fig. 1A). Alternative initiation of translation of the mRNA encoding HER2 from the methionine codons 611 and 687 generates two fragments: 611- and 687-CTF. These differ by a stretch of 76 amino acids, which includes the transmembrane domain and a cysteine-rich short extracellular domain (6) (see also Fig. 1A).Open in a separate windowFIGURE 1.Effect of HER2 CTFs on cell migration. A, schematic showing the different constructs used in these studies. The primary sequence of the extracellular and intracellular juxtamembrane domains of HER2 is shown. The transmembrane domain (TM) is represented by a hatched box. The positions of amino acids 611, 648, and 687 are indicated. The N at the beginning of the rectangle representing HER2 identifies the amino terminus. The vertical bold double line represents the plasma membrane, and the extracellular (out) and intracellular (in) regions are marked. B, MCF7 Tet-Off cells stably transfected with the empty vector (Vector) or with the vector containing the cDNAs encoding HER2 or 611-, 648-, or 687-CTFs under the control of a Tet/Dox-responsive element were kept with or without doxycycline for 24 h, lysed, and analyzed by Western blot with CB11, an antibody against the cytoplasmic domain of HER2. C, MCF7 Tet-Off cells transfected with empty vector or with 611-CTF were seeded in the absence of doxycycline, and motility was monitored by time lapse video microscopy. Representative fields of migrated cells at the indicated times are shown. Results indistinguishable from those corresponding to MCF7 Tet-Off cells transfected with vector and treated without doxycycline were observed when analyzing MCF7/611-CTF Tet-Off cells in the presence of doxycycline (data not shown). D, representative examples of the migratory behavior of MCF7 Tet-Off cells treated as in C. Digital images were taken every 30 min for 24 h (see supplemental videos), and the tracks were manually drawn. Results indistinguishable from those corresponding to MCF7 Tet-Off cells transfected with vector and treated without doxycycline were observed when analyzing MCF7/611-CTF Tet-Off cells in the presence of doxycycline (data not shown). E, tracks from cells analyzed as in D were measured in mm. Bars, average length ± S.D. of the tracks of five cells, each one on a different culture plate. F, MCF7 Tet-Off cells transfected with 611-CTF were seeded in the absence or the presence of doxycycline on transwell plates, as described under “Experimental Procedures.” After 24 h, cells were fixed and stained with DAPI. Representative fields are shown. G, MCF7 Tet-Off cells stably transfected with the empty vector (Vector) or with the vector containing the cDNAs encoding HER2 or 611-, 648-, or 687-CTFs were seeded on transwell plates with or without doxycycline as indicated. After 24 h, cells were fixed, stained with DAPI, and counted. Bars, average of the cells counted in three independent experiments, each one performed in triplicate, ± S.D. H, representative examples of the images obtained by time lapse microscopy of the closure of wounds made in a monolayer of control MCF7 Tet-Off cells stably transfected with 611-CTF and treated with or without doxycycline as indicated. The lines drawn in the images represent reference lines marking the width of the scratch when it was made. I, average values of migration distances in scratch wound assays as in H. Bars, means from three independent experiments, each one derived from evaluation of 10 fields ± S.D.We have recently shown that 687-CTF seems to be inactive (7). In contrast, the two CTFs containing the transmembrane domain, 648- and 611-CTFs, expressed at levels similar to those found in human breast tumors, can activate different intracellular signal transduction pathways (7). The level of activation of these pathways by HER2 CTFs is quite different. 611-CTF activates the mitogen-activated protein kinase and the Akt pathways to a greater extent because it constitutively forms homodimers maintained through disulfide bonds (7). In contrast, 648-CTF does not seem to form homodimers, and its activity is comparable with that of full-length HER2 (7). Therefore, cells expressing transmembrane CTFs, particularly 611-CTF, constitute a relevant model to study the consequences of the overactivation of HER2 signaling in tumors. Supporting this conclusion, it has been shown that breast cancer patients expressing CTFs have worse prognosis and are more likely to develop nodal metastasis compared with patients expressing predominantly full-length HER2 (8).Here we show that expression of 611-CTF enhances the migration of breast cancer cells as judged by monitoring single-cell migration, transwell migration, and wound healing assays. Since cell migration is frequently regulated by phosphorylation/dephosphorylation, we searched for phosphoproteins regulated by 611-CTF and probably contributing to cell migration using two independent proteomic approaches. The results of these analyses showed that difference gel electrophoresis (DIGE) is a particularly convenient methodology to analyze the regulation of multiphosphorylated proteins.Cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, was identified by DIGE as a phosphoprotein likely to be regulated by 611-CTF. Several assays showed that expression of 611-CTF leads to an increase in the phosphorylation of cortactin and to the generation of cell protrusions resembling lamellipodia or invadopodia. Confirming a role of cortactin on the increased cell migration induced by 611-CTF, down-modulation of the former with short hairpin RNAs leads to an impairment of the cell migration induced by the HER2 fragment. These results unveil a role of cortactin in the increased cell migration induced by hyperactive HER2 and strongly suggest that cortactin-dependent increased cell migration contributes to the tendency of breast tumors expressing CTFs to metastasize.     

A Mouse Model Uncovers LKB1 as an UVB-Induced DNA Damage Sensor Mediating CDKN1A (p21WAF1/CIP1) Degradation

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.     

Leptin and adiponectin, but not IL18, are related with insulin resistance in treated HIV-1-infected patients with lipodystrophy

Leptin, adiponectin and IL18 are adipokines related with obesity, insulin resistance and dyslipidemia in the general population. Treated HIV-1-infected patients with lipodystrophy may develop insulin resistance and proatherogenic dyslipidemia. We assessed the relationship between plasma adipokine levels, adipokine genetics, lipodystrophy and metabolic disturbances. Plasma leptin, adiponectin and IL18 levels were assessed in 446 individuals: 282 HIV-1-infected patients treated with antiretroviral drugs (132 with lipodystrophy and 150 without) and 164 uninfected controls (UC). The LEP2410A>G, LEPRQ223R, ADIPQ276G>T, ADIPOR2-Intron5A>G and IL18-607C>A polymorphisms were validated by sequencing. Leptin levels were higher in UC than in HIV-1-infected, either with or without lipodystrophy (p<0.001 for both comparisons) and were lower in patients with lipodystrophy compared with those without lipodystrophy (p=0.006). In patients with lipodystrophy, leptin had a positive correlation with insulin and with HOMA-IR. Adiponectin levels were non-significantly different in UC and HIV-1-infected patients. Patients with lipodystrophy had lower adiponectin levels than non-lipodystrophy subjects (p<0.001). In patients with lipodystrophy, adiponectin was negatively correlated with insulin, HOMA-IR and triglycerides. Plasma IL18 levels were higher in HIV-1-infected patients compared with UC (p<0.001), and no differences were found according to the presence of lipodystrophy. In patients with lipodystrophy there was a negative correlation between IL18 levels and LDLc. Genetic analyses indicated no significant associations with lipodystrophy nor with insulin resistance or with lipid abnormalities. In conclusion, HIV-1-infected patients have reduced plasma leptin levels. This reduction is magnified in patients with lipodystrophy whose adiponectin levels were lower than that of non-lipodystrophy subjects. Plasma IL18 levels are increased in infected patients irrespective of the presence of lipodystrophy. The polymorphisms assessed are not associated with lipodystrophy or metabolic disturbances in treated HIV-1-infected patients.     

Inactivating mutations block the tumor necrosis factor-alpha-converting enzyme in the early secretory pathway

The ectodomain of different transmembrane molecules is released by a proteolytic event known as shedding. The metalloprotease disintegrin proTNF-alpha converting enzyme (TACE) is responsible for the shedding of various proteins, including protransforming growth factor-alpha (proTGF-alpha) and amyloid-beta precursor protein (APP). Inactive TACE accumulates in the early secretory pathway of cell mutants (M1 and M2) defective in proTGF-alpha and APP shedding. Although previous evidences indicated that the component mutated in M1 and M2 cells is different from TACE, recent results show the existence of two heterozygous point mutations in TACE from M2 cells. Here, we show that wild-type TACE stably transfected in M2 cells is processed, transported to the cell surface, and rescues the proTGF-alpha and APP shedding-defective phenotype. Furthermore, M1 cells also express mutant TACE and transfection with wild-type TACE restores the wild-type phenotype. Therefore, different inactivating mutations result in the accumulation of TACE in the early secretory pathway, emphasizing the importance of the initial steps in the biosynthesis of TACE.     

Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.     

Recycling of cell surface pro-transforming growth factor-{alpha} regulates epidermal growth factor receptor activation

Impairments in signal transduction, leading to the regulation of cell proliferation, differentiation, or migration are frequently the cause of cancer. Since the accurate spatial and temporal location of their components is crucial to ensure the correct regulation of these signaling pathways, it could be anticipated that defects in intracellular trafficking are at the base of certain neoplasias. However, the trafficking of many components of pathways frequently up-regulated in cancers, such as the epidermal growth factor receptor (EGFR) pathway, are largely unknown. Here, we show that the pro-transforming growth factor-alpha (pro-TGF-alpha), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-alpha does not progress to the lysosome; instead, it is delivered to the cell surface via recycling endosomes. To analyze the functional meaning of the internalization of pro-TGF-alpha, we used a deletion construct that is normally transported to the cell surface but is deficiently endocytosed. Due to this impairment, the levels of this construct at the cell surface are dramatically augmented. Consequently, the deletion construct displays a higher EGFR-activating ability, revealing a link between the trafficking of pro-TGF-alpha and the signaling by the EGFR and opening the possibility that defects in the trafficking of the growth factor may contribute to the development of tumors.