Papers by Marianela Vara Messler
Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints, 2020
The activation of the majority of AGC kinases is regulated by two phosphorylation events on two c... more The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. this observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αc-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation. The 61 human AGC kinases form a monophyletic group of serine/threonine kinases that preferably phosphoryl-ates residues in close proximity of basic amino acids such as Arg (R) and Lys (K) 1,2. The kinase domains (KD) of all the AGC kinases share the same tertiary structure, characterized by an amino-terminal small lobe (N-lobe) and a carboxy-terminal large lobe (C-lobe), as originally described for PKA 3. The two lobes form a pocket that binds one molecule of ATP as phosphate donor during substrate phosphorylation. The transition from inactive to active state in AGC kinases is achieved through conformational rearrangements of key structural elements, such as the activation segment and the αC-helix. The activation segment is a sequence of variable length (from 25 aa of PKAa to 43 aa of MAST1) spanning from Asp-Phe-Gly (DFG) to Ala-Pro-Glu (APE) sequences, and including the activation loop (AL) and the P + 1 loop 4. The DFG sequence is part of the ATP binding site whose orientation defines the active (DFG-in) 3 and the inactive (DFG-out) states of AGC kinases 5. The AL contains, in 43 out of 61 AGC kinases (Fig. 1A), a key phosphorylatable site (consensus sequence S/T-x-x-G-T), found to be substrate of 3-Phosphoinositide-dependent protein kinase-1 (PDK1) 6. The phosphate group added on the AL form a complex set of salt bridges with basic amino acid groups, that in PKA are respectively: R165 in the catalytic loop, H87 in the αC-helix and K189 in the AL, just after the DFG motif 7,8. By connecting these residues, the phosphorylation of the AL promotes the transition in a more ordered confor-mation, the stabilization of the two lobes in the closed/active conformation and the assembly of a key hydropho-bic core, defined R-spine 9-11. Crucial event in the transition in the active conformation is the reorientation of the αC-helix 12. This event coordinates the formation of key hydrogen bonds between a Glu residue on the αC-helix, a Lys residue in the N-lobe and the phosphate of ATP, and contributes to the assembly of the R-spine 13 .
Eicosapentaenoic acid prevents salt sensitivity in diabetic rats and decreases oxidative stress, 2020
Objectives: Salt sensitivity (SS) is associated with increased cardiovascular risk in patients wi... more Objectives: Salt sensitivity (SS) is associated with increased cardiovascular risk in patients with Type 2 diabetes mellitus (T2-DM) due to an increase in renal oxidation. v-3 polyunsaturated fatty acids have shown anti-oxidant effects, but a typical Western diet contains limited content. In particular, v-3 polyunsaturated fatty acids are able to activate nuclear factor erythroid 2-related factor 2 (Nrf-2) to prevent diabetes mellitusÀ related complications by mitigating oxidative stress. Therefore, we hypothesized that eicosapentaenoic acid (EPA; v-3) modulates SS in rats with T2-DM by decreasing renal oxidative stress via Nrf-2 activation and enhancing the antiinflammatory response via interleukin (IL) 6 modulation. Methods: Three-month-old male rats (n = 40) were fed with a Normal Na-diet (NNaD) and randomly selected into four groups: Healthy Wistar nondiabetic rats (Wi), diabetic controls (eSS), arachidonic acid-treated eSS (AA; v-6), and EPA-treated eSS (v-3). After 1 year, rats were placed in metabolic cages for 7 d and fed a NNaD, followed by a 7-d period with a High Na-diet (HNaD). Systolic blood pressure, body weight, serum IL-6 and reactive oxygen species (ROS) levels were determined at the end of each 7-d period. Glycated hemoglobin (HbA1c), triacylglycerol, creatinine, and cholesterol levels were determined. ROS levels and Nrf-2 expression in kidney lysates were also assayed. Histologic changes were evaluated. A t test or analysis of variance was used for the statistical analysis. Results: After a HNaD, systolic blood pressure increased in both the control eSS and AA groups, but not in the EPA and Wi groups. However, HbA1c levels remained unchanged by the treatments, which suggests that the observed beneficial effect was independent of HbA1c levels. The IL-6 levels were higher in the eSS and AA groups, but remained unaltered in EPA and Wi rats after a HNaD diet. Interestingly, EPA protected against serum ROS in rats fed the HNaD, whereas AA did not. In kidney lysates, ROS decreased significantly in the EPA group compared with the eSS group, and Nrf-2 expression was consistently higher compared with the AA and eSS groups. Diabetic rats presented focal segmental sclerosis, adherence to Bowman capsule, and mild-to-moderate interstitial fibrosis. EPA and AA treatment prevented kidney damage. Conclusions: An adequate v3-to-v6 ratio prevents SS in diabetic rats by a mechanism that is independent of glucose metabolism but associated with the prevention of renal oxidative stress generation. These data suggest that EPA antioxidant properties may prevent the development of hypertension or kidney damage.
Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, ... more Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.
OBJECTIVE:
The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) ... more OBJECTIVE:
The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma.
MATERIALS AND METHODS:
BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma.
RESULTS:
After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERβ in tumor lysates.
CONCLUSION:
These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.
KEYWORDS:
Chia oil; Estrogen receptor; LOXs metabolites; Mammary carcinoma; α-linolenic acid (ALA)
Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFA... more Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis, and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1 and 4-1BB, in AA-treated cells. We demonstrated that downregulation of the transcription factor GLI1 in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1 and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules, and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and downregulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating NFATc1 nuclear translocation, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants reduced the AA-induced apoptosis, downregulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for the future PUFA-based therapeutic approaches.
Prostaglandins & other lipid mediators (Impact Factor: 2.86). 04/2015; DOI: 10.1016/j.prostagland... more Prostaglandins & other lipid mediators (Impact Factor: 2.86). 04/2015; DOI: 10.1016/j.prostaglandins.2015.04.003
Source: PubMed
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ABSTRACT Polyunsaturated fatty acids (PUFAs), particulary the ω-3 PUFAs and COXIBs have been associated with decreased inflammation and the prevention of tumorigenesis. ω-3 PUFAs have shown to display multiple antitumour actions, while ω-6 PUFAs and its derived eicosanoids promote the effects in cancer cell growth, angiogenesis, and invasion. ω-3 PUFAs may act by suppressing the metabolism of arachidonic acid to form proinflammatory mediators or as a precursors of novel lipid mediators with pro-resolving activity, while COXIBs are able to modulate inflammatory response by inhibiting cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase overexpressed in several human cancers. As recently has been postulated, the anti-inflammation and pro-resolution process are not equivalent. A family of lipid mediators from ω-3 PUFAs can act as agonist promoting resolution, while antinflammatory agents such as COXIBs may act as antagonists limiting the inflammatory response. The present paper reviews the current knowledge about the role of PUFAs and its derivatives (metabolites), as well as the COXIBs activity in cancer process as a sinergic therapeutic alternative for cancer treatment.
Copyright © 2015. Published by Elsevier Inc.
Urinary tract tumors are tenth in frequency, and many environmental carcinogens are excreted by u... more Urinary tract tumors are tenth in frequency, and many environmental carcinogens are excreted by urine. Interplay between chronic inflammatory urolithiasis and urothelial carcinogenesis is not well understood. Experimental evidences show that dietary melamine induce these events even at low concentrations. This is important because thousands of children were exposed to melamine through intentionally contaminated milk formula worldwide. We propose that an increased risk for urinary tumors in adult life may occur and screenings for early urinary signs may be necessary. Therefore, urothelial biology, melamine carcinogenic potential, and related epidemiology are discussed, recommending a preventive dietary polyunsaturated fatty acid-based supplementation, since they modulate such interplay in rodents.
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Papers by Marianela Vara Messler
The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma.
MATERIALS AND METHODS:
BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma.
RESULTS:
After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERβ in tumor lysates.
CONCLUSION:
These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.
KEYWORDS:
Chia oil; Estrogen receptor; LOXs metabolites; Mammary carcinoma; α-linolenic acid (ALA)
Source: PubMed
Edit
ABSTRACT Polyunsaturated fatty acids (PUFAs), particulary the ω-3 PUFAs and COXIBs have been associated with decreased inflammation and the prevention of tumorigenesis. ω-3 PUFAs have shown to display multiple antitumour actions, while ω-6 PUFAs and its derived eicosanoids promote the effects in cancer cell growth, angiogenesis, and invasion. ω-3 PUFAs may act by suppressing the metabolism of arachidonic acid to form proinflammatory mediators or as a precursors of novel lipid mediators with pro-resolving activity, while COXIBs are able to modulate inflammatory response by inhibiting cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase overexpressed in several human cancers. As recently has been postulated, the anti-inflammation and pro-resolution process are not equivalent. A family of lipid mediators from ω-3 PUFAs can act as agonist promoting resolution, while antinflammatory agents such as COXIBs may act as antagonists limiting the inflammatory response. The present paper reviews the current knowledge about the role of PUFAs and its derivatives (metabolites), as well as the COXIBs activity in cancer process as a sinergic therapeutic alternative for cancer treatment.
Copyright © 2015. Published by Elsevier Inc.
The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma.
MATERIALS AND METHODS:
BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma.
RESULTS:
After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERβ in tumor lysates.
CONCLUSION:
These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.
KEYWORDS:
Chia oil; Estrogen receptor; LOXs metabolites; Mammary carcinoma; α-linolenic acid (ALA)
Source: PubMed
Edit
ABSTRACT Polyunsaturated fatty acids (PUFAs), particulary the ω-3 PUFAs and COXIBs have been associated with decreased inflammation and the prevention of tumorigenesis. ω-3 PUFAs have shown to display multiple antitumour actions, while ω-6 PUFAs and its derived eicosanoids promote the effects in cancer cell growth, angiogenesis, and invasion. ω-3 PUFAs may act by suppressing the metabolism of arachidonic acid to form proinflammatory mediators or as a precursors of novel lipid mediators with pro-resolving activity, while COXIBs are able to modulate inflammatory response by inhibiting cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase overexpressed in several human cancers. As recently has been postulated, the anti-inflammation and pro-resolution process are not equivalent. A family of lipid mediators from ω-3 PUFAs can act as agonist promoting resolution, while antinflammatory agents such as COXIBs may act as antagonists limiting the inflammatory response. The present paper reviews the current knowledge about the role of PUFAs and its derivatives (metabolites), as well as the COXIBs activity in cancer process as a sinergic therapeutic alternative for cancer treatment.
Copyright © 2015. Published by Elsevier Inc.