Quantification of the N-glycosylated Secretome by Super-SILAC During Breast Cancer Progression and in Human Blood Samples

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines that are representative of different stages of breast cancer development by specifically capturing N-glycosylated peptides using the N-glyco FASP technology. For accurate quantification we developed a super-SILAC mix from several labeled breast cancer cell lines and used it as an internal standard for all samples. In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting that biologically relevant differences were detected. Five different profiles of glycoprotein dynamics during cancer development were detected, and they contained several proteins with known roles in breast cancer. We then used the super-SILAC mix in plasma, which led to the quantification of a large number of the previously identified N-glycopeptides in this important body fluid. The combination of quantifying the secretome of cancer cell lines and of human plasma with a super-SILAC approach appears to be a promising new approach for finding markers of disease.There has been a long-standing interest in applying proteomics to the cancer field (1). Technological advances in liquid chromatography-mass spectrometry (LC-MS) have made it feasible to profile the proteome of cancer cells to great depth (2, 3) and these developments now allow studying protein expression on a systems wide level (4). Analyses of intracellular proteins provide data on what is occurring at the intracellular level in terms of biochemical processes, signaling pathways and cellular structure. However, from a clinical perspective, focusing on proteins that are secreted by these cells is very appealing for diagnostic purposes, as they may filtrate into the peripheral blood (5). This is advantageous because peripheral blood is an easily accessible source whereas tissue biopsies are invasive and they are generally only taken when a medical condition is already suspected. Blood itself is a very complex fluid whose proteome is extremely challenging to analyze because of its very high dynamic range (6–8). Furthermore, a tumor in the initial stages would not be expected to secrete large amounts of proteins and these proteins would be severely diluted in the total blood volume (9). Therefore, discovery of biomarkers by direct analysis of blood plasma has been very difficult so far (10). A more straightforward approach would be the analysis of proteins secreted from homogeneous cell populations (11–14). Consequently, the conditioned medium of cell lines has extensively been used for the analysis of secreted cancer proteins (15). The secretome contains proteins that are actively secreted through classical and nonclassical routes but also proteins that are shed from the plasma membrane by various sheddases (12). Secretome studies are generally performed using serum-free media to reduce the initial protein contents. Further precautions are taken to minimize the contamination of intracellular proteins arising from dead cells that release their contents. Despite these caveats, the totality of proteins that are found in the conditioned medium has been referred to as the “secretome” (13).During cancer development, the invasive capacity of the cells increases progressively. Cancer cells lose cell-cell adhesion which allows eventual release of the cell from the surrounding tissue and may facilitate metastasis to other organs. The extracellular matrix is an important factor in this process as it plays a significant role in regulating numerous cellular functions like adhesion, cell shape, migration, proliferation, polarity, differentiation and apoptosis (16, 17). Many components of the extracellular matrix change in expression during cancer development. Therefore, these changes would likely be reflected in the protein contents of the secretome.Here, we set out to profile the proteins that are secreted by breast cancer cell lines from different stages by MS-based proteomics methods. For several reasons, we focused on N-glycosylated proteins as an appropriate handle to probe proteins that could be of clinical interest. First, proteins that use the classical secretion pathway or are shed from the membrane are typically N-glycosylated because they have passed through the endoplasmic reticulum (ER)¹ and Golgi system (18). Second, glycosylation may enhance the stability of the protein and protect it from proteolytic degradation (19), which would increase the likelihood of detection away from the place where the protein was produced or secreted. Third, glycosylation has a direct relationship to cancer development (20, 21). Fourth, almost all of the currently used protein biomarkers are in fact glycoproteins, such as carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and prostate-specific antigen (PSA) (22). Finally, glycoproteins have themselves been used as therapeutic targets in cancer. These include ErbB2, targeted by trastuzumab and VEGF-A, targeted by bevacizumab (23).Experimentally, a prime advantage of targeting glycosylation is the fact that glycopeptides or glycoproteins can be efficiently enriched over nonglycosylated molecules. In proteomics, enrichment targeted to N-glycosylation has typically been performed using hydrazide chemistry (24–26) or lectin based enrichment (27, 28). Our group has previously used the ‘filter aided sample preparation’ (FASP) as a basis of N-glycopeptide enrichment (29). The filter membrane in FASP can be employed to physically retain mixtures of lectins, which do not need to be coupled to beads. N-glycopeptides are first bound to the lectins and in a subsequent step simultaneously deglycosylated and released from the lectins. The complexity of the sample is thereby reduced to a level where extensive fractionation is dispensable and the highly enriched fraction of previously N-glycosylated peptides can readily be analyzed in a single high-resolution LC-MS run. We have used N-glyco-FASP to determine N-glycosylation sites in several mouse tissues (29) and in evolutionary distant model organisms (30). Here we adapted the method to supernatants of cell lines and we used the latest generation of Orbitrap analyzers for MS detection. Furthermore, to allow accurate quantification of differences in abundance levels between different secretomes, we spiked an internal standard of a super-SILAC mix (31) containing the conditioned medium of three heavy stable isotope labeled cell lines into all the conditioned medium samples. We collected the conditioned medium from a panel of eleven breast cell lines that were representative of five different cancer stages, from healthy to metastatic cells. The method was further applied to the analysis of blood plasma to verify its applicability in a body fluid context.     

Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

Objective

Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear.

Aim

The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples.

Design

In order to capture a wide range of fecal protease (FP) activity stool samples were collected from 30 IBS patients and 24 healthy controls. The intestinal microbiota was characterized using 454 high throughput pyro-sequencing of the 16S rRNA gene. The composition and diversity of microbial communities were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. FP activity levels were determined using an ELISA-based method. FP activity was ranked and top and bottom quartiles (n=13 per quartile) were identified as having high and low FP activity, respectively.

Results

The overall diversity of the intestinal microbiota displayed significant clustering separation (p = 0.001) between samples with high vs. low FP activity. The Lactobacillales, Lachnospiraceae, and Streptococcaceae groups were positively associated with FP activity across the entire study population, whilst the Ruminococcaceae family and an unclassified Coriobacteriales family were negatively associated with FP activity.

Conclusions

These data demonstrate significant associations between specific intestinal bacterial groups and fecal protease activity and provide a basis for further causative studies investigating the role of enteric microbes and GI diseases.     

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.     

In vitro and ex vivo suppression by aminoglycosides of PCDH15 nonsense mutations underlying type 1 Usher syndrome

Type 1 Usher syndrome (USH1) is a recessively inherited condition, characterized by profound prelingual deafness, vestibular areflexia, and prepubertal onset of retinitis pigmentosa (RP). While the auditory component of USH1 can be treated by cochlear implants, to date there is no effective treatment for RP. USH1 can be caused by mutations in each of at least six genes. While truncating mutations of these genes cause USH1, some missense mutations of the same genes cause nonsyndromic deafness. These observations suggest that partial or low level activity of the encoded proteins may be sufficient for normal retinal function, although not for normal hearing. In individuals with USH1 due to nonsense mutations, interventions enabling partial translation of a full-length functional protein may delay the onset and/or progression of RP. One such possible therapeutic approach is suppression of nonsense mutations by small molecules such as aminoglycosides. We decided to test this approach as a potential therapy for RP in USH1 patients due to nonsense mutations. We initially focused on nonsense mutations of the PCDH15 gene, underlying USH1F. Here, we show suppression of several PCDH15 nonsense mutations, both in vitro and ex vivo. Suppression was achieved both by commercial aminoglycosides and by NB30, a new aminoglycoside-derivative developed by us. NB30 has reduced cytotoxicity in comparison to commercial aminoglycosides, and thus may be more efficiently used for therapeutic purposes. The research described here has important implications for the development of targeted interventions that are effective for patients with USH1 caused by various nonsense mutations. Annie Rebibo-Sabbah and Igor Nudelman contributed equally to this work.     

Structure-altering mutations of the SARS-CoV-2 frameshifting RNA element

With the rapid rate of COVID-19 infections and deaths, treatments and cures besides hand washing, social distancing, masks, isolation, and quarantines are urgently needed. The treatments and vaccines rely on the basic biophysics of the complex viral apparatus. Although proteins are serving as main drug and vaccine targets, therapeutic approaches targeting the 30,000 nucleotide RNA viral genome form important complementary approaches. Indeed, the high conservation of the viral genome, its close evolutionary relationship to other viruses, and the rise of gene editing and RNA-based vaccines all argue for a focus on the RNA agent itself. One of the key steps in the viral replication cycle inside host cells is the ribosomal frameshifting required for translation of overlapping open reading frames. The RNA frameshifting element (FSE), one of three highly conserved regions of coronaviruses, is believed to include a pseudoknot considered essential for this ribosomal switching. In this work, we apply our graph-theory-based framework for representing RNA secondary structures, “RAG (or RNA-As-Graphs),” to alter key structural features of the FSE of the SARS-CoV-2 virus. Specifically, using RAG machinery of genetic algorithms for inverse folding adapted for RNA structures with pseudoknots, we computationally predict minimal mutations that destroy a structurally important stem and/or the pseudoknot of the FSE, potentially dismantling the virus against translation of the polyproteins. Our microsecond molecular dynamics simulations of mutant structures indicate relatively stable secondary structures. These findings not only advance our computational design of RNAs containing pseudoknots, they pinpoint key residues of the SARS-CoV-2 virus as targets for antiviral drugs and gene editing approaches.     

The little fire ant Wasmannia auropunctata: a new invasive species in the Middle East and its impact on the local arthropod fauna

The little fire ant, Wasmannia auropunctata, probably arrived in Israel in ca. 1998 and was identified in 2005; this is the first record of this species from open areas outside the tropics and subtropics. It survives harsher conditions than in its native habitats, with minimal annual temperatures as low as 6°C, and 5–12 consecutive rainless months (under 15 mm rainfall per month). It is now known from 26 localities in Israel, mostly in irrigated gardens. As in other regions where they have invaded, these ants pose a serious threat to local biodiversity. At high densities they displaced almost all the local ant species sampled, affecting population abundances, species richness, and community structure. W. auropunctata seems to have a detrimental effect also on other ground arthropods, judging from the observed decline in spider and beetle abundances. We show here that this tropical species can pose a critical threat to local arthropods at a wider range of climatic conditions than was previously known.