Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions.

Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.

Proteomic analyses of brain tumor cell lines amidst the unfolded protein response.

Brain tumors such as high grade gliomas are among the deadliest forms of human cancers. The tumor environment is subject to a number of cellular stressors such as hypoxia and glucose deprivation. The persistence of the stressors activates the unfolded proteins response (UPR) and results in global alterations in transcriptional and translational activity of the cell. Although the UPR is known to effect tumorigenesis in some epithelial cancers, relatively little is known about the role of the UPR in brain tumors. Here, we evaluated the changes at the molecular level under homeostatic and stress conditions in two glioma cell lines of differing tumor grade. Using mass spectrometry analysis, we identified proteins unique to each condition (unstressed/stressed) and within each cell line (U87MG and UPN933). Comparing the two, we find differences between both the conditions and cell lines indicating a unique profile for each. Finally, we used our proteomic data to identify the predominant pathways within these cells under unstressed and stressed conditions. Numerous predominant pathways are the same in both cell lines, but there are differences in biological and molecular classifications of the identified proteins, including signaling mechanisms, following UPR induction; we see that relatively minimal proteomic alterations can lead to signaling changes that ultimately promote cell survival.

Potential Transfer of Polyglutamine and CAG-Repeat RNA in Extracellular Vesicles in Huntington's Disease: Background and Evaluation in Cell Culture.

In Huntington's disease (HD) the imperfect expanded CAG repeat in the first exon of the HTT gene leads to the generation of a polyglutamine (polyQ) protein, which has some neuronal toxicity, potentially mollified by formation of aggregates. Accumulated research, reviewed here, implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in extracellular vesicles (EVs). Most cell types in the brain release EVs and these have been shown to contain neurodegenerative proteins in the case of prion protein and amyloid-beta peptide. In this study, we used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of Hdh mRNA containing the expanded repeat incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another, but further work will be needed to evaluate potential EV and cell-type specificity of transfer and effects of long-term exposure. It seems likely that expanded HD-associated repeat RNA may appear in biofluids and may have use as biomarkers of disease state and response to therapy.

Glioma-derived extracellular vesicles selectively suppress immune responses.

BACKGROUND: Glioma-related immunosuppression is well documented; however, the mechanisms of suppression are not fully understood. Here we explore a role for glioma extracellular vesicles (EVs) as a means of immune modulation. METHODS: Healthy donor peripheral blood mononuclear cells (PBMCs) were incubated with mitogenic stimuli and various concentrations of glioma-derived EVs. Intracellular signaling and cytokine output were determined by protein microarrays, and phenotypic changes were assessed by flow cytometry. Recall antigen testing, mixed lymphocyte reactions, and migration assays analyzed PBMC functional capacity. RESULTS: Protein microarray data revealed induction of an immunosuppressive phenotype and cytokine output at high tumor-vesicle concentrations but an activated phenotype at low concentrations. T cell activation antigen expression confirmed differential activation profiles. Functional analyses revealed decreased migratory capacity of PBMCs after incubation with EVs; however, recall antigen and mixed lymphocyte tests indicated that activation capacity is still retained in EV-treated cells. CONCLUSION: The differential effects of high and low EV concentrations dictate modulatory effects on PBMCs. These data provide a role for EVs at high concentrations for inducing selective tolerance of an immune response in a tumor setting. This suggests that lymphocytes in patients' circulation are not irreparably impaired, as previously thought, but can be rescued to augment antitumor responses.

Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro.

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over "immortalized" cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (>10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.

Extracellular RNA mediates and marks cancer progression.

Different types of RNAs identified thus far represent a diverse group of macromolecules that are involved in the regulation of different biological processes. RNA is generally thought to be localized primarily in the nucleus and cytoplasm; however, some types of RNA have been detected in the extracellular milieu. These extracellular RNA (exRNA) molecules are protected from degradation and it is now widely accepted that extracellular vesicles and ribonucleoprotein particles serve as transport vehicles for exRNA among cells. The functional consequence of this transfer of genetic information probably encompasses a broad range of normal developmental and physiologic processes in many organisms. This review will focus on the role of exRNA communication in cancer. We will focus on different types of RNA species identified and characterized within tumor-derived extracellular vesicles. Further, we will describe the role of exRNAs in cancer progression, as well as their potential for use as diagnostic biomarkers and therapeutic tools for monitoring and treating cancer, respectively.

Glioblastoma extracellular vesicles: reservoirs of potential biomarkers.

Glioblastoma multiforme (GBM) is the most frequent and most devastating of the primary central nervous system tumors, with few patients living beyond 2 years postdiagnosis. The damage caused by the disease and our treatments for the patients often leave them physically and cognitively debilitated. Generally, GBMs appear after very short clinical histories and are discovered by imaging (using magnetic resonance imaging [MRI]), and the diagnosis is validated by pathology, following surgical resection. The treatment response and diagnosis of tumor recurrence are also tracked by MRI, but there are numerous problems encountered with these monitoring modalities, such as ambiguous interpretation and forms of pseudoprogression. Diagnostic, prognostic, and predictive biomarkers would be an immense boon in following treatment schemes and in determining recurrence, which often requires an invasive intracranial biopsy to verify imaging data. Extracellular vesicles (EVs) are stable, membrane-enclosed, virus-sized particles released from either the cell surface or from endosomal pathways that lead to the systemic release of EVs into accessible biofluids, such as serum/plasma, urine, cerebrospinal fluid, and saliva. EVs carry a wide variety of proteins, nucleic acids, lipids, and other metabolites, with many common features but with enough individuality to be able to identify the cell of origin of the vesicles. These components, if properly interrogated, could allow for the identification of tumor-derived EVs in biofluids, indicating tumor progression, relapse, or treatment failure. That knowledge would allow clinicians to continue with treatment regimens that were actually effective or to change course if the therapies were failing. Here, we review the features of GBM EVs, in terms of EV content and activities that may lead to the use of EVs as serially accessible biomarkers for diagnosis and treatment response in neuro-oncology.

Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-ß1 and EMMPRIN.

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-ß1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity.

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. CD147 expression is so high in several cancers that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2, which is ubiquitously expressed in most tissues, and CD147 Ig0-Ig1-Ig2, which is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins, both with and without the CD147 Ig0 domain, within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.

Proteome and system ontology of hemorrhagic shock: exploring early constitutive changes in postshock mesenteric lymph.

BACKGROUND: Postshock mesenteric lymph (PSML) is the mechanistic link between splanchnic ischemia reperfusion (IR) and remote organ injury. We hypothesize that an unbiased inspection of the proteome of PSML will reveal previously unrecognized aberrations in systems biology provoked by hemorrhage-induced mesenteric IR injury in vivo. METHODS: Shock was induced in male Sprague-Dawley rats by controlled hemorrhage, and the mesenteric duct was cannulated for lymph collection. Preshock and postshock lymph were collected for differential in-gel electrophoresis (DIGE)-based proteomics. Proteins that increased or decreased in relative concentration > or =1.5-fold were selected for trypsin digestion and analysis by mass spectrometry (MS). RESULTS: Evidence of tissue injury was detected by an increase in cell/tissue proteins in PSML. Components of coagulation were depleted, whereas products of hemolysis were increased. Haptoglobin was decreased, which supports an early postshock hemolytic process. Interestingly, several protective protease inhibitors were decreased in PSML. The unexpected findings were an increase in alpha-enolase (a key glycolitic enzyme and cell-surface plasminogen binding receptor, +2.4-fold change) and increased major urinary protein (MUP, a sex-specific lipid-binding protein, +17.1-fold change) in PSML. CONCLUSION: A proteomic evaluation of PSML revealed evidence of several shock-associated processes: protein release from tissue injury, depletion of coagulation factors and evidence of hemolysis, depletion of protective protease inhibitors, and an increase in abundance of lipid carriers. These results suggest that constitutive changes in the proteome of PSML may provide novel insights into the complex pathophysiology of postshock systems biology.