The Structural Proteins of Membrane Rafts, Caveolins and Flotillins, in Lung Cancer: More Than Just Scaffold Elements.

Lung cancer is one of the most frequently diagnosed cancers worldwide. Due to its late diagnosis, it remains the leading cause of cancer-related deaths. Despite it is mostly associated to tobacco smoking, recent data suggested that genetic factors are of the highest importance. In this context, different processes meaningful for the development and progression of lung cancer such endocytosis, protein secretion and signal transduction, are controlled by membrane rafts. These highly ordered membrane domains contain proteins such as caveolins and flotillins, which were traditionally considered scaffold proteins but have currently been given a preponderant role in lung cancer. Here, we summarize current knowledge regarding the involvement of caveolins and flotillins in lung cancer from a molecular point of view.

On The Role of Myelin and Lymphocyte Protein (MAL) In Cancer: A Puzzle With Two Faces.

Myelin and lymphocyte protein (MAL) is an integral membrane protein constituent of lipid rafts, and it is implicated in apical transport of proteins in polarized epithelial cells. However, beyond the involvement of MAL in apical sorting and as its function as a raft stabilizer, it is still not totally clear how MAL participates in cell proliferating processes. More controversial and interesting is the fact that MAL has been implicated in carcinogenesis in two opposite ways. First, this protein is overexpressed in ovarian cancer and some kinds of lymphomas where it seems to favor cancer progression. Conversely, it has been reported that downregulation of the MAL gene by promoter hypermethylation is a hallmark of several adenocarcinomas. So far, there is not enough experimental evidence to help us understand this phenomenon, and no MAL mutations or MAL isoforms have been associated with these opposite functions. This review provides an updated summary of the structure and functions of MAL, and we will discuss the possible mechanisms underlying its roles as a tumor suppressor and a tumor progression factor.

The mitochondrial O-linked N-acetylglucosamine transferase (mOGT) in the diabetic patient could be the initial trigger to develop Alzheimer disease.

Diabetes mellitus (DM) is considered a risk factor for the development of Alzheimer disease (AD); however, how DM favors evolution of AD is still insufficiently understood. Hyperglycemia in DM is associated to an increase in mitochondrial reactive oxygen species (ROS) generation, as well as damage of hippocampal cells, reflected by changes in morphological and mitochondrial functionality. Similar mitochondrial damage has been observed when amyloid beta (Aß) accumulates in the brain of AD patients. In DM, the excess of glucose in the brain induces higher activity of the hexosamine biosynthesis pathway (HBP), it synthesizes UDP-N-acetylglucosamine (UDP-GlcNAc), which is used by O-linked N-acetylglucosamine transferase (OGT) to catalyze O-GlcNAcylation of numerous proteins. Although O-GlcNAcylation plays an important role in maintaining structure and cellular functionality, chronic activity of this pathway has been associated with insulin resistance and hyperglycemia-induced glucose toxicity. Three different forms of OGT are known: nucleocytoplasmic (ncOGT), short (sOGT), and mitochondrial (mOGT). Previous reports showed that overexpression of ncOGT is not toxic to the cell; in contrast, overexpression of mOGT is associated with cellular apoptosis. In this work, we suggest that hyperglycemia in the diabetic patient could induce greater expression and activity of mOGT, modifying the structure and functionality of mitochondria in hippocampal cells, accelerating neuronal damage, and favoring the start of AD. In consequence, mOGT activity could be a key point for AD development in patients with DM.

Inefficient translocation of preproinsulin contributes to pancreatic ß cell failure and late-onset diabetes.

Among the defects in the early events of insulin biosynthesis, proinsulin misfolding and endoplasmic reticulum (ER) stress have drawn increasing attention as causes of ß cell failure. However, no studies have yet addressed potential defects at the cytosolic entry point of preproinsulin into the secretory pathway. Here, we provide the first evidence that inefficient translocation of preproinsulin (caused by loss of a positive charge in the n region of its signal sequence) contributes to ß cell failure and diabetes. Specifically, we find that, after targeting to the ER membrane, preproinsulin signal peptide (SP) mutants associated with autosomal dominant late-onset diabetes fail to be fully translocated across the ER membrane. The newly synthesized, untranslocated preproinsulin remains strongly associated with the ER membrane, exposing its proinsulin moiety to the cytosol. Rather than accumulating in the ER and inducing ER stress, untranslocated preproinsulin accumulates in a juxtanuclear compartment distinct from the Golgi complex, induces the expression of heat shock protein 70 (HSP70), and promotes ß cell death. Restoring an N-terminal positive charge to the mutant preproinsulin SP significantly improves the translocation defect. These findings not only reveal a novel molecular pathogenesis of ß cell failure and diabetes but also provide the first evidence of the physiological and pathological significance of the SP n region positive charge of secretory proteins.

Effect of Histoplasma capsulatum glucans on host innate immunity / Efecto de los glucanos de Histoplasma capsulatum sobre la inmunidad innata del huésped

Beta-1,3-Glucan is important for infective forms (mycelial phase) of Histoplasma capsulatum and shares many features allotted to pathogen-associated molecular patterns. These cell wall carbohydrates interact with phagocytes by binding to Toll and lectin-like receptors, present on cell surfaces of macrophages, neutrophils, and dendritic cells. This review focuses on recent findings of the major H. capsulatum and host carbohydrate-driven interactions that account for internalization of fungal infective forms into phagocytes, and its subsequent avoidance of intracellular elimination. The yeast phase of H. capsulatum possesses different modulating factors of the macrophagic-anti-fungal mechanisms, mainly alpha-1,3-glucan, which is considered relevant for virulence. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

El Beta-1,3-glucano es importante para las formas infectivas (fase micelial) de Histoplasma capsulatum y comparte varias características asignadas a los patrones moleculares asociados con patógenos. Estos hidratos de carbono de la pared celular interaccionan con los fagocitos uniéndose a receptores tipo Toll y tipo lectina, que están presentes en las superficies celulares de macrófagos, neutrófilos y células dendríticas. En esta revisión se presta atención a los hallazgos recientes sobre las principales interacciones entre H. capsulatum y las células del huésped mediadas por hidratos de carbono, que permiten la internalización de las formas infectivas del hongo por los fagocitos, así como la posterior evitación de su eliminación intracelular. Se discuten los datos experimentales relevantes publicados recientemente. La fase de levadura de H. capsulatum incluye distintos factores moduladores de los mecanismos de macrófagos y antifúngicos, sobre todo el alpha-1,3-glucano, que se considera relevante para la virulencia.Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Effect of Histoplasma capsulatum glucans on host innate immunity.

ß-1,3-Glucan is important for infective forms (mycelial phase) of Histoplasma capsulatum and shares many features allotted to pathogen-associated molecular patterns. These cell wall carbohydrates interact with phagocytes by binding to Toll and lectin-like receptors, present on cell surfaces of macrophages, neutrophils, and dendritic cells. This review focuses on recent findings of the major H. capsulatum and host carbohydrate-driven interactions that account for internalization of fungal infective forms into phagocytes, and its subsequent avoidance of intracellular elimination. The yeast phase of H. capsulatum possesses different modulating factors of the macrophagic-anti-fungal mechanisms, mainly α-1,3-glucan, which is considered relevant for virulence. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Impaired cleavage of preproinsulin signal peptide linked to autosomal-dominant diabetes.

Recently, missense mutations upstream of preproinsulin's signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying ß-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER-oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY.

Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth.

Type 1B diabetes (typically with early onset and without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene Induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed mutant proinsulins exerting dominant-negative (toxic gain of function) behavior in pancreatic beta cells. Here we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that, in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.

Insulin storage and glucose homeostasis in mice null for the granule zinc transporter ZnT8 and studies of the type 2 diabetes-associated variants.

OBJECTIVE: Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS: Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS: ZnT8(-/-) mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS: ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

Endoplasmic reticulum (ER) chaperone regulation and survival of cells compensating for deficiency in the ER stress response kinase, PERK.

The activity of PERK, an endoplasmic reticulum (ER) transmembrane protein kinase, assists in an ER stress response designed to inhibit general protein synthesis while allowing upregulated synthesis of selective proteins such as the ATF4 transcription factor. PERK null mice exhibit phenotypes that especially affect secretory cell types. Although embryonic fibroblasts from these mice are difficult to transfect with high efficiency, we have generated 293 cells stably expressing the PERK-K618A dominant negative mutant. 293/PERK-K618A cells, in response to ER stress: (a) do not properly inhibit general protein synthesis, (b) exhibit defective/delayed induction of ATF4 and BiP, and (c) exhibit exuberant splice activation of XBP1 and robust cleavage activation of ATF6, with abnormal regulation of calreticulin levels. The data suggest compensatory mechanisms allowing for cell survival in the absence of functional PERK. Interestingly, although induction of CHOP (a transcription factor implicated in apoptosis) is notably delayed after onset of ER stress, 293/PERK-K618A cells eventually produce CHOP at normal or even supranormal levels and exhibit increased apoptosis either in response to general ER stress or, more importantly, to specific misfolded secretory proteins.

Dominant-negative behavior of mammalian Vps35 in yeast requires a conserved PRLYL motif involved in retromer assembly.

The retromer protein complex assists in recycling selected integral membrane proteins from endosomes to the trans Golgi network. One protein subcomplex (Vps35p, Vps26p and Vps29p) combines with a second (Vps17p and Vps5p) to form a coat involved in sorting and budding of endosomal vesicles. Yeast Vps35p (yVps35) exhibits similarity to human Vps35 (hVps35), especially in a completely conserved PRLYL motif contained within an amino-terminal domain. Companion studies indicate that an R(98)W mutation in yVps35 causes defective retromer assembly in Saccharomyces cerevisiae. Herein, we find that the expression of hVps35 in yeast confers dominant-negative vacuolar proenzyme secretion and defective secretory proprotein processing. The mutant phenotype appears to be driven by hVps35 competing with endogenous yVps35, becoming incorporated into defective retromer complexes and causing proteasomal degradation of endogenous Vps26 and Vps29. Increased expression of yVps35 displaces some hVps35 to a 100 000 x g supernatant and suppresses the dominant-negative phenotype. Remarkably, mutation of the conserved R(107)W of hVps35 displaces some of the protein to the 100 000 x g supernatant, slows protein turnover and restores stability of Vps26p and Vps29p and completely abrogates dominant-negative trafficking behavior. We show that hVps35 coprecipitates Vps26, whereas the R(107)W mutant does not. In pancreatic beta cells, the R(107)W mutant shifts hVps35 from peripheral endosomes to a juxtanuclear compartment, affecting both mannose phosphate receptors and insulin. These data underscore importance of the Vps35 PRLYL motif in retromer subcomplex interactions and function.

Lumenal protein sorting to the constitutive secretory pathway of a regulated secretory cell.

Newly synthesized secretory granule content proteins are delivered via the Golgi complex for storage within mature granules, whereas constitutive secretory proteins are not stored. Most soluble proteins traveling anterograde through the trans-Golgi network are not excluded from entering immature secretory granules, whether or not they have granule-targeting signals. However, the ;sorting-for-entry' hypothesis suggests that soluble lumenal proteins lacking signals enter transport intermediates for the constitutive secretory pathway. We aimed to investigate how these constitutive secretory proteins are sorted. In a pancreatic beta-cell line, we stably expressed two lumenal proteins whose normal sorting information has been deleted: alkaline phosphatase, truncated to eliminate its glycosylphosphatidylinositol membrane anchor (SEAP); and Cab45361, a Golgi lumenal resident, truncated to eliminate its intracellular retention (Cab308Myc). Both truncated proteins are efficiently secreted, but whereas SEAP enters secretory granules, Cab308Myc behaves as a true constitutive marker excluded from granules. Interestingly, upon permeabilization of organelle membranes with saponin, SEAP is extracted as a soluble protein whereas Cab308Myc remains associated with the membrane. These are among the first data to support a model in which association with the lumenal aspect of Golgi and/or post-Golgi membranes can serve as a means for selective sorting of constitutive secretory proteins.