Victor García-González

Protein Folding and Mechanisms of Proteostasis

Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis.

Disorder-to-order conformational transitions in protein structure and its relationship to disease

Function in proteins largely depends on the acquisition of specific structures through folding at physiological time scales. Under both equilibrium and non-equilibrium states, proteins develop partially structured molecules that being intermediates in the process, usually resemble the structure of the fully folded protein. These intermediates, known as molten globules, present the faculty of adopting a large variety of conformations mainly supported by changes in their side chains. Taking into account that the mechanism to obtain a fully packed structure is considered more difficult energetically than forming partially “disordered” folding intermediates, evolution might have conferred upon an important number of proteins the capability to first partially fold and—depending on the presence of specific partner ligands—switch on disorder-to-order transitions to adopt a highly ordered well-folded state and reach the lowest energy conformation possible. Disorder in this context can represent segments of proteins or complete proteins that might exist in the native state. Moreover, because this type of disorder-to-order transition in proteins has been found to be reversible, it has been frequently associated with important signaling events in the cell. Due to the central role of this phenomenon in cell biology, protein misfolding and aberrant disorder-to-order transitions have been at present associated with an important number of diseases.

Reality of a Vaccine in the Prevention and Treatment of Atherosclerosis

Atherosclerosis together with multiple sclerosis, psoriasis and rheumatoid arthritis can be used as examples of chronic inflammatory diseases associated with multifactorial components that evolve over the years. Nevertheless, an important difference between these diseases relies on the fact that atherosclerosis develops from early ages where inflammation dominates the very beginning of the disease. This review highlights the inflammatory nature of atherosclerosis and the role the immune system plays in the process of atherogenesis. Although treatment of atherosclerosis has been for years based on lipid-lowering therapies reducing a series of risk factors, the degree of success has been only limited because cardiovascular complications related to the evolution of atherosclerotic lesions continue to appear in the population worldwide. In this sense, alternative treatments for atherosclerosis have come into play where both innate and adaptive immunity have been proposed to modulate atherosclerosis-associated inflammatory phenomena. When tested for their atheroprotective properties, several immunogens have been studied through passive and active immunization with good results and, therefore, the strategy through vaccination to control the disease has been made possible. Many experimental pre-clinical studies demonstrating proof of concept that vaccination using DNA and protein with an effective use of adjuvants and the optimal route of administration now provide a tangible new therapeutic approach that sets the stage for several of these vaccines to be tested in large, randomized, long-term clinical studies. A vaccine ready for human use will only be accomplished through the close association between academia, regulatory government organizations and private industry, allowing the reality of a simple and successful therapy to reduce atherosclerosis and its severe clinical complications.

Key structural arrangements at the C-terminus domain of CETP suggest a potential mechanism for lipid-transfer activity.

The cholesteryl-ester transfer protein (CETP) promotes cholesteryl-ester and triglyceride transfer between lipoproteins. We evaluated the secondary structure stability of a series of small peptides derived from the C-terminus of CETP in a wide range of pHs and lipid mixtures, and studied their capability to carry out disorder-to-order secondary structure transitions dependent of lipids. We report that while a mixture of phosphatidylcholine/cholesteryl-esters forms large aggregated particles, the inclusion of a series of CETP carboxy-terminal peptides in a stable α-helix conformation, allows the formation of small homogeneous micelle-like structures. This phenomenon of lipid ordering was directly connected to secondary structural transitions at the C-terminus domain when lysophosphatidic acid and lysophosphatidylcholine lipids were employed. Circular dichroism, cosedimentation experiments, electron microscopy, as well as molecular dynamics simulations confirm this phenomenon. When purified CETP is studied, the same type of phenomenon occurs by promoting the reorganization of lipid from large to smaller particles. Our findings extend the emerging view for a novel mechanism of lipid transfer carried out by CETP, assigning its C-terminus domain the property to accomplish lipid ordering through secondary structure disorder-to-order transitions.

Amyloidogenic properties of a D/N mutated 12 amino acid fragment of the C-terminal domain of the Cholesteryl-Ester Transfer Protein (CETP).

The cholesteryl-ester transfer protein (CETP) facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D470N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS) and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when β-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D470N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D470N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of amyloid fibrils in the highly flexible C-terminus domain of CETP.

The C-terminal Domain Supports a Novel Function for CETPI as a New Plasma Lipopolysaccharide-Binding Protein.

Described by our group a few years ago, the cholesteryl-ester transfer protein isoform (CETPI), exclusively expressed in the small intestine and present in human plasma, lacked a functional identification for a role of physiological relevance. Now, this study introduces CETPI as a new protein with the potential capability to recognise, bind and neutralise lipopolysaccharides (LPS). Peptides derived from the C-terminal domain of CETPI showed that CETPI not only might interact with several LPS serotypes but also might displace LPS bound to the surface of cells. Peptide VSAK, derived from the last 18 residues of CETPI, protected against the cytotoxic effect of LPS on macrophages. At high concentrations, when different cell types were tested in culture, it did not exhibit cytotoxicity by itself and it did prevent the expression of pro-inflammatory cytokines as well as the generation of oxidative stress conditions. In a rabbit model of septic shock, the infusion of peptide VSAK exerted a protective effect against the effects of LPS and reduced the presence of tumor necrosis factor-alpha (TNFα) in plasma. Therefore, CETPI is proposed as a new protein with the capability to advance the possibilities for better understanding and treatment of the dangerous effects of LPS in vivo.

A Novel β-adaptin/c-Myc Complex Formation Modulated by Oxidative Stress in the Control of the Cell Cycle in Macrophages and its Implication in Atherogenesis

Our study tested the proposal that c-Myc activation in macrophages is differentially carried out dependent on the intracellular oxidative state of cells and potentially associated to the process of atherogenesis. Under our experimental conditions, the generation of reactive oxygen species carried out by the presence of oxidized low density lipoproteins (oxLDL) or Gram negative bacterial lipopolysaccharides (LPS) modifies the expression of cellular adhesion molecules such as c-Abl, calcium transport proteins such as the plasma membrane Ca2+-ATPase (PMCA), CD47, procaspase-7, CASP7, CHOP, transcriptional activators such as c-Jun and c-Myc and molecules that participate in the process of endocytosis like α- and β-adaptin. We present the first evidence showing that a state of oxidative stress alters c-Myc-dependent activity pathways in macrophages through binding to molecules such as β-adaptin promoting the reversible formation of a complex that presents the ability to regulate the development of the cell cycle. We propose that the subtle regulation carried out through the formation of this c-Myc/β-adaptin complex when cells change from a normal physiological condition to a state of oxidative stress, represents a defense mechanism against the deleterious effects caused by the loss of cell homeostasis.

Modulation of Amyloidogenesis Controlled by the C-Terminal Domain of Islet Amyloid Polypeptide Shows New Functions on Hepatocyte Cholesterol Metabolism

The islet amyloid polypeptide (IAPP) or amylin maintains a key role in metabolism. This 37-residues-peptide could form pancreatic amyloids, which are a characteristic feature of diabetes mellitus type 2. However, some species do not form amyloid fibril structures. By employing a biomimetic approach, we generated an extensive panel of optimized sequences of IAPP, which could drastically reduce aggregation propensity. A structural and cellular characterization analysis was performed on the C-terminal domain with the highest aggregation propensity. This allowed the observation of an aggregative phenomenon dependent of the lipid environment. Evaluation of the new F23R variant demonstrated inhibition of β-sheet structure and, therefore, amyloid formation on the native C-terminal, phenomenon that was associated with functional optimization in calcium and cholesterol management coupled with the optimization of insulin secretion by beta cells. When F23R variant was evaluated in microglia cells, a model of amyloidosis, cytotoxic conditions were not registered. In addition, it was found that C-terminal sequences of IAPP could modulate cholesterol metabolism in hepatocytes through regulation of SREBP-2, apoA-1, ABCA1, and LDLR, mechanism that may represent a new function of IAPP on the metabolism of cholesterol, increasing the LDL endocytosis in hepatocytes. Optimized sequences with only one residue modification in the C-terminal core aggregation could diminish β-sheet formation and represent a novel strategy adaptable to other pharmacological targets. Our data suggest a new IAPP function associated with rearrangements on metabolism of cholesterol in hepatocytes.

Protein translation associated to PERK arm is a new target for regulation of metainflammation: A connection with hepatocyte cholesterol: GALINDO-HERNÁNDEZ et al.

Endoplasmic reticulum stress is a cellular phenomenon that has been associated with metabolic disorders, contributing to the development of obesity, fatty liver disease, and dyslipidemias. Under metabolic overload conditions, in cells with a high protein‐secretory activity, such as hepatocytes and Langerhans β cells, the unfolded protein response (UPR) is critical in to maintain protein homeostasis (proteostasis). UPR integrated by a tripartite signaling system, through activating transcription factor 6, protein kinase R‐like endoplasmic reticulum kinase (PERK), and inositol‐requiring enzyme 1, regulates gene transcription and translation to resolve stress and conserve proteostasis. In the current study, we demonstrated in hepatocytes under metabolic overload by saturated palmitic and stearic fatty acids, through activation of PERK signaling and CCAAT‐enhancer‐binding protein homologous protein (CHOP) transcription factor, an association with the expression of cyclooxygenase 2. More important, isolated exosomes from supernatants of macrophages exposed to lipopolysaccharides can also induce a metainflammation phenomenon, and when treated on hepatocytes, induced a rearrangement in cholesterol metabolism through sterol regulatory element‐binding protein 2 (SREBP2), low‐density lipoprotein receptor (LDLR), apolipoprotein A‐I, and ABCA1. Moreover, we demonstrate the cellular effect of terpene‐derived molecules, such as cryptotanshinone, isolated of plant Salvia brandegeei, regulating metainflammatory conditions through PERK pathway in both hepatocytes and β cells. Our data suggest the presence of a modulatory mechanism on specific protein translation process. This effect could be mediated by eukaryotic initiation factor‐4A, evaluating salubrinal as a control molecule. Likewise, the protective mechanisms of unsaturated fatty acids, such as oleic and palmitoleic acid were confirmed. Therefore, modulation of metainflammation suggests a new target through PERK signaling in cells with a high secretory activity, and possibly the regulation of cholesterol in hepatocytes is promoted via exosomes.