Mechanisms by which obesity regulates inflammation and anti-tumor immunity in cancer.

Obesity is associated with an increased risk for 13 different cancers. The increased risk for cancer in obesity is mediated by obesity-associated changes in the immune system. Obesity has distinct effects on different types of inflammation that are tied to tumorigenesis. For example, obesity promotes chronic inflammation in adipose tissue that is tumor-promoting in peripheral tissues. Conversely, obesity inhibits acute inflammation that rejects tumors. Obesity therefore promotes cancer by differentially regulating chronic versus acute inflammation. Given that obesity is chronic, the initial inflammation in adipose tissue will lead to systemic inflammation that could induce compensatory anti-inflammatory reactions in peripheral tissues to suppress chronic inflammation. The overall effect of obesity in peripheral tissues is therefore dependent on the duration and severity of obesity. Adipose tissue is a complex tissue that is composed of many cell types in addition to adipocytes. Further, adipose tissue cellularity is different at different anatomical sites throughout the body. Consequently, the sensitivity of adipose tissue to obesity is dependent on the anatomical location of the adipose depot. For example, obesity induces more inflammation in visceral than subcutaneous adipose tissue. Based on these studies, the mechanisms by which obesity promotes tumorigenesis are multifactorial and immune cell type-specific. The objective of our paper is to discuss the cellular mechanisms by which obesity promotes tumorigenesis by regulating distinct types of inflammation in adipose tissue and the tumor microenvironment.

mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

Antioxidant Activity during Tumor Progression: A Necessity for the Survival of Cancer Cells?

Antioxidant defenses encompass a variety of distinct compounds and enzymes that are linked together through their capacity to neutralize and scavenge reactive oxygen species (ROS). While the relationship between ROS and tumorigenesis is clearly complex and context dependent, a number of recent studies have suggested that neutralizing ROS can facilitate tumor progression and metastasis in multiple cancer types through distinct mechanisms. These studies therefore infer that antioxidant activity may be necessary to support the viability and/or the invasive capacity of cancer cells during tumor progression and metastasis. Here, we discuss some of the accumulating evidence suggesting a role for antioxidant activity in facilitating tumor progression.

Characterization of the binding between a 70-kDa heat shock protein, HspA1A, and phosphoinositides.

HspA1A, a seventy-kilodalton heat shock protein, binds to specific anionic lipids and this interaction regulates important physiological phenomena like apoptosis, tumor growth, and lysosomal rescue. However, whether HspA1A binds to phosphoinositides has yet to be established and quantified. Therefore, in this study, we determined the binding affinity of HspA1A to several phosphoinositides and characterized five aspects of their molecular interaction. First, we established that HspA1A binds phosphatidylinositol monophosphates with higher affinity than di- and triphosphorylated inositides. Second, using high concentrations of potassium we found that HSPA1A embeds within the lipid bilayer of all phosphoinositides tested. However, the effects of the high salt concentrations were significantly different between the different phosphoinositides. Third, using calcium and reaction buffers equilibrated at different pH values we found that these differentially affected HspA1A-phosphoinositide binding, revealing a lipid-specific pattern of binding. Fourth, by assessing the binding properties of the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, we determined that in most cases the full-length protein is necessary for binding to phosphoinositides. Fifth, by including in the reactions nucleotides and protein substrates we determined that they minimally and differentially affected phosphoinositide-binding. Collectively, these findings strongly suggest that the HspA1A-phosphoinositide binding is complex yet specific, is mediated by both electrostatic and hydrophobic interactions, is not related to the lipid-head charge, and depends on the physicochemical properties of the lipid.

Biochemical characterization of the interaction between HspA1A and phospholipids.

Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.

HspA1A, a 70-kDa heat shock protein, differentially interacts with anionic lipids.

HspA1A, a 70-kDa heat shock protein, binds to specific lipids. This interaction allows HspA1A to associate with the plasma and other cellular membranes, where it regulates many vital functions like immunity, membrane stabilization, autophagy, and apoptosis. However, the molecular mechanism of the HspA1A-lipid interactions has yet to be fully characterized. Therefore, in this study, we characterized the interaction of HspA1A with three lipids, bis-(monoacylglycero)-phosphate, cardiolipin, and sulfatide. Our results revealed that, first, HspA1A embeds in membranes when bound to liposomes composed of cardiolipin and sulfatide. Second, the binding of HspA1A to lipids is complex and although important, electrostatic interactions alone cannot fully explain the observed binding. Third, the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, differentially bind to lipids in a lipid-specific manner. Fourth, HspA1A lipid-binding is reduced by the presence of nucleotides, but it is unaffected by the presence of a peptide-substrate. These observations suggest that HspA1A binds to lipids via a multi-step mechanism and this interaction depends on the specific physicochemical properties of the lipid. We speculate that the association of HspA1A with lipids like the mitochondrial cardiolipin, which is an organelle marker, may facilitate the translocation and localized function of the molecular chaperone to particular sub-cellular compartments.

Functional diversification and specialization of cytosolic 70-kDa heat shock proteins.

A fundamental question in molecular evolution is how protein functional differentiation alters the ability of cells and organisms to cope with stress and survive. To answer this question we used two paralogous Hsp70s from mouse and explored whether these highly similar cytosolic molecular chaperones, which apart their temporal expression have been considered functionally interchangeable, are differentiated with respect to their lipid-binding function. We demonstrate that the two proteins bind to diverse lipids with different affinities and therefore are functionally specialized. The observed lipid-binding patterns may be related with the ability of both Hsp70s to induce cell death by binding to a particular plasma-membrane lipid, and the potential of only one of them to promote cell survival by binding to a specific lysosomal-membrane lipid. These observations reveal that two seemingly identical proteins differentially modulate cellular adaptation and survival by having acquired specialized functions via sequence divergence. Therefore, this study provides an evolutionary paradigm, where promiscuity, specificity, sub- and neo-functionalization orchestrate one of the most conserved systems in nature, the cellular stress-response.

Sequence variation of koala retrovirus transmembrane protein p15E among koalas from different geographic regions.

The koala retrovirus (KoRV), which is transitioning from an exogenous to an endogenous form, has been associated with high mortality in koalas. For other retroviruses, the envelope protein p15E has been considered a candidate for vaccine development. We therefore examined proviral sequence variation of KoRV p15E in a captive Queensland and three wild southern Australian koalas. We generated 163 sequences with intact open reading frames, which grouped into 39 distinct haplotypes. Sixteen distinct haplotypes comprising 139 of the sequences (85%) coded for the same polypeptide. Among the remaining 23 haplotypes, 22 were detected only once among the sequences, and each had 1 or 2 non-synonymous differences from the majority sequence. Several analyses suggested that p15E was under purifying selection. Important epitopes and domains were highly conserved across the p15E sequences and in previously reported exogenous KoRVs. Overall, these results support the potential use of p15E for KoRV vaccine development.

Evolutionary genomics of immunoglobulin-encoding Loci in vertebrates.

Immunoglobulins (or antibodies) are an essential element of the jawed vertebrate adaptive immune response system. These molecules have evolved over the past 500 million years and generated highly specialized proteins that recognize an extraordinarily large number of diverse substances, collectively known as antigens. During vertebrate evolution the diversification of the immunoglobulin-encoding loci resulted in differences in the genomic organization, gene content, and ratio of functional genes and pseudogenes. The tinkering process in the immunoglobulin-encoding loci often gave rise to lineage-specific characteristics that were formed by selection to increase species adaptation and fitness. Immunoglobulin loci and their encoded antibodies have been shaped repeatedly by contrasting evolutionary forces, either to conserve the prototypic structure and mechanism of action or to generate alternative and diversified structures and modes of function. Moreover, evolution favored the development of multiple mechanisms of primary and secondary antibody diversification, which are used by different species to effectively generate an almost infinite collection of diverse antibody types. This review summarizes our current knowledge on the genomics and evolution of the immunoglobulin-encoding loci and their protein products in jawed vertebrates.

Comparative genomics and evolution of immunoglobulin-encoding loci in tetrapods.

The immunoglobulins (Igs or antibodies) as an integral part of the tetrapod adaptive immune response system have evolved toward producing highly diversified molecules that recognize a remarkably large number of different antigens. Antibodies and their respective encoding loci have been shaped by different and often contrasting evolutionary forces, some of which aim to conserve an established pattern or mechanism and others to generate alternative and diversified structural and functional configurations. The genomic organization, gene content, ratio between functional genes and pseudogenes, number and position of recombining genetic elements, and the different levels of divergence present at the germline of the Ig-encoding loci have been evolutionarily shaped and optimized in a lineage- and, in some cases, species-specific mode aiming to increase organismal fitness. Further, evolution favored the development of multiple mechanisms of primary and secondary antibody diversification, such as V(D)J recombination, class switch recombination, isotype exclusion, somatic hypermutation, and gene conversion. Diverse tetrapod species, based on their specific germline configurations, use these mechanisms in several different combinations to effectively generate a vast array of distinct antibody types and structures. This chapter summarizes our current knowledge on the Ig-encoding loci in tetrapods and discusses the different evolutionary mechanisms that shaped their diversification.

Comparative genomics and evolution of the alpha-defensin multigene family in primates.

Defensin genes encode small cationic antimicrobial peptides that form an important part of the innate immune system. They are divided into three families, alpha (α), beta (ß), and theta (), according to arrangement of the disulfide bonding pattern between cysteine residues. Considering the functional importance of defensins, investigators have studied the evolution and the genomic organization of defensin genes. However, these studies have been restricted mainly to ß-defensins. To understand the evolutionary dynamics of α-defensin genes among primates, we identified the α-defensin repertoires in human, chimpanzee, orangutan, macaque, and marmoset. The α-defensin genes in primates can be classified into three phylogenetic classes (class I, II, and III). The presence of all three classes in the marmoset indicates that their divergence occurred before the separation of New World and Old World monkeys. Comparative analysis of the α-defensin genomic clusters suggests that the makeup of the α-defensin gene repertoires between primates is quite different, as their genes have undergone dramatic birth-and-death evolution. Analysis of the encoded peptides of the α-defensin genes indicates that despite the overall high level of sequence divergence, certain amino acid residues or motifs are conserved within and between the three phylogenetic classes. The evolution of α-defensins in primates, therefore, appears to be governed by two opposing evolutionary forces. One force stabilizes specific amino acid residues and motifs to preserve the functional and structural integrity of the molecules and the other diversifies the sequences generating molecules with a wide range of activities against a large number of pathogens.