Cross-platform proteomics signatures of extreme old age.

In previous work we used a Somalogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals.

Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, ß1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that ß1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low ß1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed ß1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

Protein signatures of centenarians and their offspring suggest centenarians age slower than other humans.

Using samples from the New England Centenarian Study (NECS), we sought to characterize the serum proteome of 77 centenarians, 82 centenarians' offspring, and 65 age-matched controls of the offspring (mean ages: 105, 80, and 79 years). We identified 1312 proteins that significantly differ between centenarians and their offspring and controls (FDR < 1%), and two different protein signatures that predict longer survival in centenarians and in younger people. By comparing the centenarian signature with 2 independent proteomic studies of aging, we replicated the association of 484 proteins of aging and we identified two serum protein signatures that are specific of extreme old age. The data suggest that centenarians acquire similar aging signatures as seen in younger cohorts that have short survival periods, suggesting that they do not escape normal aging markers, but rather acquire them much later than usual. For example, centenarian signatures are significantly enriched for senescence-associated secretory phenotypes, consistent with those seen with younger aged individuals, and from this finding, we provide a new list of serum proteins that can be used to measure cellular senescence. Protein co-expression network analysis suggests that a small number of biological drivers may regulate aging and extreme longevity, and that changes in gene regulation may be important to reach extreme old age. This centenarian study thus provides additional signatures that can be used to measure aging and provides specific circulating biomarkers of healthy aging and longevity, suggesting potential mechanisms that could help prolong health and support longevity.

MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth.

Intrinsically disordered proteins (IDPs)/intrinsically unstructured proteins are characterized by the lack of fixed or stable tertiary structure, and are increasingly recognized as an important class of proteins with major roles in signal transduction and transcriptional regulation. In this study, we report the identification and functional characterization of a previously uncharacterized protein (UPF0258/KIAA1024), major intrinsically disordered Notch2-associated receptor 1 (MINAR1). While MINAR1 carries a single transmembrane domain and a short cytoplasmic domain, it has a large extracellular domain that shares no similarity with known protein sequences. Uncharacteristically, MINAR1 is a highly IDP with nearly 70% of its amino acids sequences unstructured. We demonstrate that MINAR1 physically interacts with Notch2 and its binding to Notch2 increases its stability and function. MINAR1 is widely expressed in various tissues including the epithelial cells of the breast and endothelial cells of blood vessels. MINAR1 plays a negative role in angiogenesis as it inhibits angiogenesis in cell culture and in mouse matrigel plug and zebrafish angiogenesis models. Furthermore, while MINAR1 is highly expressed in the normal human breast, its expression is significantly downregulated in advanced human breast cancer and its re-expression in breast cancer cells inhibited tumor growth. Our study demonstrates that MINAR1 is an IDP that negatively regulates angiogenesis and growth of breast cancer cells.

Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation.

Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed.

Semi-automated identification of N-Glycopeptides by hydrophilic interaction chromatography, nano-reverse-phase LC-MS/MS, and glycan database search.

Glycoproteins fulfill many indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. Improved analytical methods are needed to allow a complete characterization of this complex and common post-translational modification. In this study, we present a workflow for the analysis of the microheterogeneity of N-glycoproteins that couples hydrophilic interaction and nanoreverse-phase C18 chromatography to tandem QTOF mass spectrometric analysis. A glycan database search program, GlycoPeptideSearch, was developed to match N-glycopeptide MS/MS spectra with the glycopeptides comprised of a glycan drawn from the GlycomeDB glycan structure database and a peptide from a user-specified set of potentially glycosylated peptides. Application of the workflow to human haptoglobin and hemopexin, two microheterogeneous N-glycoproteins, identified a total of 57 distinct site-specific glycoforms in the case of haptoglobin and 14 site-specific glycoforms of hemopexin. Using glycan oxonium ions and peptide-characteristic glycopeptide fragment ions and by collapsing topologically redundant glycans, the search software was able to make unique N-glycopeptide assignments for 51% of assigned spectra, with the remaining assignments primarily representing isobaric topological rearrangements. The optimized workflow, coupled with GlycoPeptideSearch, is expected to make high-throughput semiautomated glycopeptide identification feasible for a wide range of users.

Cadmium effects on transcriptional expression of rhlB/rhlC genes and congener distribution of monorhamnolipid and dirhamnolipid in Pseudomonas aeruginosa IGB83.

While variable production of the biosurfactant, rhamnolipid, by Pseudomonas aeruginosa has been shown to be dependent on growth conditions, no research has evaluated potential relationships between rhamnolipid production and the presence of heavy metals. The current investigation evaluates the influence of Cd(2+) on rhamnolipid synthesis. Cultures grown in the presence of 0.45 and 0.89 mM Cd(2+) were monitored for rhlB/rhlC expression, rhamnolipid yield, and the ratio of monorhamnolipid (RL1) and dirhamnolipid (RL2) produced. Results show a Cd-induced enhancement of rhlB expression in mid-stationary phase (53 h). In addition, sustained production of rhamnolipid through late stationary growth phase (96 h) was observed for Cd-amended cultures, unlike Cd-free control cultures that ceased rhamnolipid production by mid-stationary growth phase. Most significant was an observed increase in the ratio of RL2 to RL1 congeners produced by cultures grown in the presence of Cd(2+). Previous results have shown that the complexation constant for RL2-Cd is several orders of magnitude larger than that of RL1-Cd thus the preferential production of RL2 in the presence of Cd(2+) impacts its bioavailability and toxicity both for the cell and in the surrounding environment.

Novel selective human melanocortin-3 receptor ligands: use of the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold.

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH(2)) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH(2)) by replacing the His-d-Phe and His-d-Nal(2') fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx=D-Phe/pCl-D-Phe/D-Nal(2')). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists.

Development of cyclic gamma-MSH analogues with selective hMC3R agonist and hMC3R/hMC5R antagonist activities.

A series of cyclic lactam analogues of gamma-MSH (H-Tyr1-Val2-Met3-Gly4-His5-Phe6-Arg7-Trp8-Asp9-Arg10-Phe11-Gly12-OH) with a bulky hydrophobic residue in the direct proximity to the pharmacophore (Xaa-D-Phe/D-Nal(2')-Arg-Trp) were designed and synthesized by solid-phase methods. A variety of amino acids with a broad range of hydrophobic/hydrophilic properties was introduced in position 5 to further explore their complementary role in receptor selectivity. Biological evaluation of these peptides revealed several analogues with potent hMC3R agonist and hMC3R/hMC5R antagonist activities, and good receptor selectivity. Analogue 4, c[Nle-Arg-D-Phe-Arg-Trp-Glu]-NH2, was found to be a very potent and selective hMC3R agonist (EC50=1.2 nM, 112% act). In addition, analogue 13, c[Nle-Val-D-Nal(2')-Arg-Trp-Glu]-NH2, was identified as an hMC3R/hMC5R antagonist with the best selectivity against the hMC4R in this series (pA2(hMC3R)=8.4; pA2(hMC5R)=8.7). These results indicate the significance of steric factors in melanocortin receptor selectivity and suggest that introduction of bulky residues in the direct proximity to the melanocortin pharmacophore is an effective approach to design of novel hMC3R and hMC5R selective ligands.