Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture

The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis.In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.The two defining characteristics of human embryonic stem cells (hESCs),¹ self-renewal and pluripotency, are maintained by a delicate balance of intracellular and extracellular signaling processes. Extracellular regulation is primarily the result of changes in the microenvironment surrounding the cells during growth in vitro or in vivo. HESCs interact with this “niche ” through support cells, extracellular matrix (ECM) components, and autocrine/paracrine signaling (reviewed in Refs. 1–3). Modulation of any of these supportive elements individually or in combination has been used extensively to alter hESC behavior (1–3).The culture of hESCs, as well as that of human induced pluripotent stem cells (hiPSCs), is conventionally performed on a layer of irradiated mouse embryonic fibroblast cells (MEFs). These MEFs are believed to promote the maintenance of hESCs and hiPSCs through the secretion of beneficial support proteins and cytokines into the soluble microenvironment. A number of proteomic studies have been conducted that examine the secretome of feeder-cell layers in an attempt to elucidate proteins and pathways essential for hESC and hiPSC survival (4–7). Alternatively, hESCs and hiPSCs can be cultured in feeder-free conditions in the absence of support cells. In feeder-free conditions, hESCs and hiPSCs are most often grown on the basement membrane matrix Matrigel™ in medium that has been previously conditioned by MEFs (MEF-CM). Matrigel™ is a gelatinous mixture that is secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (8). Although recent studies have proposed that a variety of defined matrices can support the growth of hESCs and hiPSCs, few of these can maintain a wide range of stem cell lines and therefore are typically not used in place of Matrigel™. The properties of Matrigel™ that make it such an effective matrix for hESC and hiPSC culture remain poorly understood. Because of the complexity of matrices like Matrigel™, the majority of proteomic studies that examine the hESC and hiPSC microenvironment have focused on contributions from support cells and soluble extracellular factors.The ECM is typically a complex network of structural proteins and glycosaminoglycans that function to support cells through the regulation of processes such as adhesion and growth factor signaling (9). Thus, it is not surprising that the generation of a well-defined matrix capable of facilitating hESC and hiPSC self-renewal has remained difficult (10). Previous proteomic investigations of Matrigel™ and other matrices supportive of hESC maintenance in vitro have revealed the presence of numerous growth, binding, and signaling proteins (11, 12). Further examination of how hESCs and hiPSCs interact with these complex matrices would provide critical information about what role the ECM plays in the organization of processes involved in the regulation of self-renewal and pluripotency.A recent study has established the ability of hESC-derived matrix microenvironments to alter tumorigenic properties through the reprogramming of metastatic melanoma cells (13). Importantly, this effect was found to be dependent on the exposure of metastatic cells to hESC-derived conditioned Matrigel™. Culture of metastatic melanoma cells in hESC-conditioned medium did not promote the reprogramming effect. These data suggest that the proteins responsible for this effect were integrated in the matrix. With the use of immunochemical techniques, it was later found that the left-right determination (Lefty) proteins A and B that were deposited in the matrix by hESCs during conditioning were at least in part responsible for the cellular change observed in metastatic cells (14). The Lefty A and B proteins are antagonists of transforming growth factor (TGF)-β signaling that act directly on Nodal protein, a critical regulator of the stem cell phenotype (15, 16). Subsequent studies of conditioned matrix utilizing mESCs implicated the bone morphogenic protein (BMP) 4 antagonist Gremlin as a primary regulator of the observed changes in metastatic cells (17). Collectively, these studies were all biased by a targeted analysis of potential effectors of metastatic cells. A comprehensive proteomic analysis of conditioned matrix could potentially reveal other factors involved in metastatic cell reprogramming. Furthermore, proteomic examination of hESC and hiPSC conditioned matrix could expose factors important in the regulation of self-renewal and pluripotency by the microenvironment in vitro.To this end, we have analyzed both types of human pluripotent stem cells, hESCs and hiPSCs, via a mass spectrometry (MS)-based proteomics approach to identify proteins deposited during growth in feeder-free conditions in vitro on Matrigel™. To investigate the hESC- and hiPSC-derived matrix, the metabolic labeling technique known as stable isotope labeling with amino acids in cell culture (SILAC) was used (18). SILAC facilitates the identification of hESC- and hiPSC-derived proteins that would otherwise be confounded by the presence of mouse-derived protein background from Matrigel™. From the proteomic analysis of three cells lines, namely, the hESC lines H9 and CA1 and the hiPSC line BJ-1D, we identified a total of 621, 1355, and 1350 total unique proteins, respectively. This work represents the first analysis of a hESC- and hiPSC-derived conditioned matrix and resulted in the identification of at least one novel microenvironmental contributor responsible for the regulation of human pluripotent stem cells.     

Comprehensive Quantitative Comparison of the Membrane Proteome,Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process.Pluripotent embryonic stem cell (ESC)¹-derived neural stem cells (NSCs) can differentiate into neurons and glia cells of the central nervous system (1), including specialized neuron types like dopaminergic, representing a potential source for treatment of neurological diseases, such as Parkinson′s disease. Therefore, a better understanding of the cellular processes behind the changes of hESCs into NSCs, including solid markers for each cell type, is fundamental to move forward with a successful regenerative cell therapy and to investigate the early human neurogenesis processes.Many markers have been reported for the two types of stem cells (2, 3), however several of these markers are also identified in other stem or progenitor cells such as CD133 (Prominin-1) (4). Discovery of cell surface specific markers for differentiated stem cells is highly relevant for future clinical applications. In particular being able to distinguish the developmental stages of the differentiation from parental stem cells to fully mature cells would allow a correct manipulation and isolation of the cell type of interest. Moreover, such study would increase our understanding on the whole process of differentiation from embryonic cells to neural cells. Because plasma membrane-associated proteins are the key interface between cell and the surrounding environment, and they frequently present large extracellular domains suitable for antibody detection, they represent a great marker candidate potential. In addition, these proteins are very important in the cellular signaling process and cell–cell interaction and communication, processes very important for cellular differentiation. Furthermore, most membrane bound proteins involved in the abovementioned process are frequently regulated or otherwise manipulated to alter interaction partners and function by post-translational modifications (PTMs) such as phosphorylation and glycosylation.Protein phosphorylation and glycosylation are the most common PTMs in nature and they play an important role in many protein regulatory functions and cellular and biological processes. Protein phosphorylation is a dynamic PTM involved in many different cell signaling events like cell cycle, protein synthesis, protein degradation, differentiation, as well cellular proliferation and apoptosis (5). On the other hand, protein glycosylation has several roles in cell–cell interaction, cell-matrix interaction, communication and cellular signaling (6–8). A nine carbon sugar unit termed sialic acid (SA) can be bound to the nonreducing ends of glycans attached to certain membrane proteins, secreted proteins and lipids (9). SA is involved in many physiological processes such as cell–cell interaction and molecular recognition, and is important in different pathophysiological process, including brain development and cancer metastasis (10–14).The mass spectrometry-based proteomic approach is a powerful tool for characterization of proteins and their PTMs. Because of the low abundance of protein PTMs in comparison to their nonmodified counterpart in the cell or tissue, their detection and characterization are almost only possible by using advanced enrichment strategies. Although many studies have reported the proteome and phosphoproteome of hESCs and early differentiated stages (15–20), there are a limited number of studies available comparing the individual stages between hESCs and NSC (21, 22) especially at the PTM level. For example, Chaerkady and collaborators have reported the quantitative temporal proteomic analysis of hESC differentiation into neural cell types, including motor neurons and astrocytes (21). The authors identified a total of 1251 proteins including proteins differentially regulated during neural differentiation such as the solute carrier protein 3 member 2 (SLC3A2), a cell surface protein highly expressed only in the hESCs stage. However, the focus of this study was not on membrane bound proteins and the authors did not analyze any PTM profile during differentiation, which may also elicit important and more selective information regarding the molecular events underlying the different stages.To obtain more information about membrane proteins involved in the changes of hESCs to NSCs and also to investigate potential markers for the two distinct cellular stages, we performed a comprehensive quantitative mass-spectrometry-based proteome and PTM-ome study of membrane fractions isolated from hESCs and NSCs. We focused the PTM study on phosphorylation and SA N-linked glycosylation. This study allowed us to identify several significantly regulated proteins in hESCs and NSCs, including proteins involved in the early embryonic development as well as in the neural development. In the latter group of proteins we could identify Crumbs homolog 2 (CR2) as a potential novel NSC marker. By using selective reaction monitoring (SRM) we were able to verify a number of potential markers including CRB2 at protein and PTM level as well as CMP-N-acetylneuraminate-poly-alpha-2,8-sialyltransferase (SIA8D) at the PTM level across different cell lines beside the one used in this study. In addition, calmodulin-dependent protein kinase-2 (CaMKII) could be an important kinase for the NSC stage, because we identified the sequence recognition motif (R-X-X-pS/T) highly up-regulated in these cells. This is the consensus site for several kinases including CaMKII. Moreover, the analysis of the regulated dataset revealed an over-representation of the extracellular matrix (ECM)-receptor pathways, which are involved in diverse processes such as differentiation and proliferation (23). To our knowledge this comparative proteome study of the membrane-associated proteins that include quantitative analysis of protein phosphorylation and SA N-linked glycosylation is the most diverse set of PTMs reported for hESCs and NSCs.     

Candidate Serological Biomarkers for Cancer Identified from the Secretomes of 23 Cancer Cell Lines and the Human Protein Atlas

Although cancer cell secretome profiling is a promising strategy used to identify potential body fluid-accessible cancer biomarkers, questions remain regarding the depth to which the cancer cell secretome can be mined and the efficiency with which researchers can select useful candidates from the growing list of identified proteins. Therefore, we analyzed the secretomes of 23 human cancer cell lines derived from 11 cancer types using one-dimensional SDS-PAGE and nano-LC-MS/MS performed on an LTQ-Orbitrap mass spectrometer to generate a more comprehensive cancer cell secretome. A total of 31,180 proteins was detected, accounting for 4,584 non-redundant proteins, with an average of 1,300 proteins identified per cell line. Using protein secretion-predictive algorithms, 55.8% of the proteins appeared to be released or shed from cells. The identified proteins were selected as potential marker candidates according to three strategies: (i) proteins apparently secreted by one cancer type but not by others (cancer type-specific marker candidates), (ii) proteins released by most cancer cell lines (pan-cancer marker candidates), and (iii) proteins putatively linked to cancer-relevant pathways. We then examined protein expression profiles in the Human Protein Atlas to identify biomarker candidates that were simultaneously detected in the secretomes and highly expressed in cancer tissues. This analysis yielded 6–137 marker candidates selective for each tumor type and 94 potential pan-cancer markers. Among these, we selectively validated monocyte differentiation antigen CD14 (for liver cancer), stromal cell-derived factor 1 (for lung cancer), and cathepsin L1 and interferon-induced 17-kDa protein (for nasopharyngeal carcinoma) as potential serological cancer markers. In summary, the proteins identified from the secretomes of 23 cancer cell lines and the Human Protein Atlas represent a focused reservoir of potential cancer biomarkers.Cancer is a major cause of mortality worldwide, accounting for 10 million new cases and more than 6 million deaths per year. In developing countries, cancer is the second most common cause of death, accounting for 23–25% of the overall mortality rate (1). Notwithstanding improvements in diagnostic imaging technologies and medical treatments, the long term survival of most cancer patients is poor. Cancer therapy is often challenging because the majority of cancers are initially diagnosed in their advanced stages. For example, the 5-year survival rate for patients with HNC¹ is less than 50%. More than 50% of all HNC patients have advanced disease at the time of diagnosis (2, 3). Enormous effort has been devoted to screening and characterizing cancer markers for the early detection of cancer. Thus far, these markers include carcinoembryonic antigen, prostate-specific antigen, α-fetoprotein, CA 125, CA 15-3, and CA 19-9. Unfortunately, most biomarkers have limited specificity, sensitivity, or both (4). Thus, there is a growing consensus that marker panels, which are more sensitive and specific than individual markers, would increase the efficacy and accuracy of early stage cancer detection (4–8). The development of novel and useful biomarker panels is therefore an urgent need in the field of cancer management.Proteomics technology platforms are promising tools for the discovery of new cancer biomarkers (9). Over the past decade, serum and plasma have been the major targets of proteomics studies aimed at identifying potential cancer biomarkers (10–13). However, the progress of these studies has been hampered by the complex nature of serum/plasma samples and the large dynamic range between the concentrations of different proteins (14). As cancer biomarkers are likely to be present in low amounts in blood samples, the direct isolation of these markers from plasma and serum samples requires a labor-intensive process involving the depletion of abundant proteins and extensive protein fractionation prior to mass spectrometric analysis (15–18). Alternatively, the secretome, or group of proteins secreted by cancer cells (19), can be analyzed to identify circulating molecules present at elevated levels in serum or plasma samples from cancer patients. These proteins have the potential to act as cancer-derived marker candidates, which are distinct from host-responsive marker candidates. We, along with other groups, have demonstrated the efficacy of secretome-based strategies in a variety of cancer types, including NPC (20), breast cancer (21, 22), lung cancer (23, 24), CRC (25, 26), oral cancer (27), prostate cancer (28, 29), ovarian cancer (30), and Hodgkin lymphoma (31). In these studies, proteins secreted from cancer cells into serum-free media were resolved by one- or two-dimensional gels followed by in-gel tryptic digestion and analysis via MALDI-TOF MS or LC-MS/MS. Alternatively, the proteins were trypsin-digested in solution and analyzed by LC-MS/MS. In general, more proteins were detected in the secretome using the LC-MS/MS method than the MALDI-TOF MS method. Advanced protein separation and identification technologies have made it possible to detect more proteins in the secretomes of cancer cells, thereby facilitating the discovery of cancer biomarkers.Although the cancer cell secretomes of various tumor types have been individually analyzed by different groups using distinct protocols, few studies have used the same protocol to compare cancer cell secretomes derived from different tumor types. We previously assessed the secretomes of 21 cancer cell lines derived from 12 cancer types (i.e. consisting of 795 protein identities and 325 non-redundant proteins) by one-dimensional gel and MALDI-TOF MS (25). Our preliminary findings revealed that different cell lines have distinct secreted protein profiles and that several putative biomarkers, such as Mac-2BP (20, 26, 27, 29) and cathepsin D (21, 23, 32), present in the secretome of a given cancer cell type are commonly shared among different cancers. These observations suggest that an in-depth comparison of secretomes derived from different tumor types may identify marker candidates common to most cancers as well as markers for specific cancer types. As an increasing number of proteins are identified in the secretomes of various cancer cell lines, scientists are faced with the challenge of quickly and efficiently narrowing down the list to candidates with higher chances of success during validation testing with precious clinical specimens.In the present study, we applied one-dimensional SDS-PAGE in conjunction with nano-LC-MS/MS (GeLC-MS/MS) (33, 34) to analyze the conditioned media of 23 cancer cell lines derived from 11 cancer types, including NPC, breast cancer, bladder cancer, cervical cancer, CRC, epidermoid carcinoma, liver cancer, lung cancer, T cell lymphoma, oral cancer, and pancreatic cancer. Within this data set, 4,584 non-redundant proteins were identified from a total of 23 cell lines, yielding an average of ∼1,300 proteins per cell line. Potential marker candidates were identified via the comparative analysis of different cell line secretomes and by putative linkages to cancer-relevant pathways. The selected proteins were further compared with the HPA (35) to generate a focused data set of proteins that are secreted or released, cancer type-specific, and highly expressed in human cancer tissues. Finally, we selectively validated four proteins as potential serological cancer markers using blood samples from cancer patients.     

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.     

Differential Processing of Amyloid-�� Precursor Protein Directs Human Embryonic Stem Cell Proliferation and Differentiation into Neuronal Precursor Cells

The amyloid-β precursor protein (AβPP) is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-β (Aβ) protein, is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease, Down syndrome, and head injury. We recently reported that this protein, normally associated with neurodegenerative conditions, is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AβPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-β, which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AβPP cleavage by β-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression, an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AβPPα, which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AβPP is normally required for embryonic neurogenesis.The amyloid-β precursor protein (AβPP)⁵ is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-β (Aβ) protein, is deposited in amyloid plaques in the aged brain, following head injury, and in the neurodegenerative conditions of Alzheimer disease (AD) and Down syndrome (DS). AβPP has structural similarity to growth factors (1) and modulates several important neurotrophic functions, including neuritogenesis, synaptogenesis, and synaptic plasticity (2). The function of AβPP during early embryogenesis and neurogenesis has not been well described.AβPP is processed by at least two pathways, the non-amyloidogenic and amyloidogenic pathways. Non-amyloidogenic processing of AβPP yields secreted AβPPα (sAβPPα), the secreted extracellular domain of AβPP that acts as a growth factor for many cell types and promotes neuritogenesis (3). Amyloidogenic processing of AβPP releases sAβPPβ, the AβPP intracellular domain, and Aβ proteins. The Aβ protein has both neurotoxic and neurotrophic properties (4) dependent on the differentiation state of the neuron; Aβ is neurotoxic to differentiating neurons via a mechanism involving differentiation-associated increases in the phosphorylation of the microtubule-associated protein tau (5) but neurotrophic to undifferentiated embryonic neurons. Evidence supporting a neurotrophic function for Aβ during development include its neurogenic activity toward rat neural stem cells (4–6). Consistent with these data, two studies have demonstrated increased hippocampal neurogenesis in young transgenic mice overexpressing human APP_Sw,Ind (7, 8).Recently we reported that human embryonic stem cells (hESCs) express AβPP and that both the stemness of the cells and the pregnancy-associated hormone human chorionic gonadotropin alter AβPP expression (9). These results suggest a functional role for AβPP during early human embryogenesis. To further investigate the function of AβPP and its cleavage products during early embryonic neurogenesis, we examined the expression and processing of this protein and its role in proliferation and differentiation of hESCs into neural precursor cells (NPCs). We found that amyloidogenic processing of AβPP promotes hESC proliferation whereas non-amyloidogenic processing induces hESC differentiation into NPCs. These data reveal an important function for AβPP during early human embryonic neurogenesis. Our data imply that any dysregulation in AβPP processing that leads to altered sAβPPα/Aβ production could result in aberrant neurogenesis as reported in the AD and DS brains.     

Secretomic Analysis Identifies Alpha-1 Antitrypsin (A1AT) as a Required Protein in Cancer Cell Migration,Invasion, and Pericellular Fibronectin Assembly for Facilitating Lung Colonization of Lung Adenocarcinoma Cells

Metastasis is a major obstacle that must be overcome for the successful treatment of lung cancer. Proteins secreted by cancer cells may facilitate the progression of metastasis, particularly within the phases of migration and invasion. To discover metastasis-promoting secretory proteins within cancer cells, we used the label-free quantitative proteomics approach and compared the secretomes from the lung adenocarcinoma cell lines CL1-0 and CL1-5, which exhibit low and high metastatic properties, respectively. By employing quantitative analyses, we identified 660 proteins, 68 of which were considered to be expressed at different levels between the two cell lines. High levels of A1AT were secreted by CL1-5, and the roles of A1AT in the influence of lung adenocarcinoma metastasis were investigated. Molecular and pathological confirmation demonstrated that altered expression of A1AT correlates with the metastatic potential of lung adenocarcinoma. The migration and invasion properties of CL1-5 cells were significantly diminished by reducing the expression and secretion of their A1AT proteins. Conversely, the migration and invasion properties of CL1-0 cells were significantly increased through the overexpression and secretion of A1AT proteins. Furthermore, the assembly levels of the metastasis-promoting pericellular fibronectin (FN1), which facilitates colonization of lung capillary endothelia by adhering to the cell surface receptor dipeptidyl peptidase IV (DPP IV), were higher on the surfaces of suspended CL1-5 cells than on those of the CL1-0 cells. This discovery reflects previous findings in breast cancer. In line with this finding, FN1 assembly and the lung colonization of suspended CL1-5 cells were inhibited when endogenous A1AT protein was knocked down using siRNA. The major thrust of this study is to demonstrate the effects of coupling the label-free proteomics strategy with the secretomes of cancer cells that differentially exhibit invasive and metastatic properties. This provides a new opportunity for the effective identification of metastasis-associated proteins that are secreted by cancer cells and promote experimental metastasis.Lung cancer is the leading cause of cancer death, and ∼90% of all lung cancer deaths are attributed to metastases (1). Approximately 95% of lung cancer patients are not diagnosed until they develop symptoms, and 85% of the newly diagnosed lung cancer patients are already in the advanced stages of the disease (2, 3). Once the tumor cells have metastasized and spread throughout the lungs, the cancer is considerably more difficult to treat. Invasiveness and metastasis are major threats to successful treatment. Cancer metastasis is an intricate, multi-step process in which the tumor cells must gain both migratory and invasive properties (4). In metastasis research, there are two common in vivo models, spontaneous and experimental metastasis (5–7). In brief, spontaneous metastasis refers to primary tumor cells that are able to dissociate from the primary tumor and metastasize to the secondary organ via the circulatory system. In contrast, experimental metastasis refers to the injection of tumor cells directly into the systemic circulation. Many researchers have attempted to determine the molecular basis of these transitions in hopes of developing target-specific drugs or biomarkers for the prevention and diagnosis of metastasis. Although there have been many discoveries regarding a particular protein''s influence on metastasis, the contribution of many protein targets to the metastatic process remains poorly defined.The term “secretome” was originally coined to refer to the secretory proteins from the entire genome of Bacillus subtilis (8). The word secretome has developed a broader meaning and now refers to the proteins released by a cell, tissue, or organism through various mechanisms, which include classical secretion, nonclassical secretion, membrane protein shedding, and secretion via exosomes (9–11). Each step involved in tumor metastasis, including migration and invasion, requires specific molecular interactions by both the tumor cells and the surrounding extracellular matrix (12). Some interactions are mediated by secretory factors that function as catalytic agents or by specific recognitions. For example, cathepsins, a family of lysosomal cysteine and aspartic proteases, plays a role in breaking down the connective barriers in the extracellular matrix and basement membranes, effectively enhancing the metastasis of tumor cells (13). These unique functions correlate with invasive activity and are otherwise known as the promigratory and pro-invasive effects on cells (14, 15). With respect to cancer progression, chronic changes or abnormal secretions of certain proteins may indicate a pathologic condition and, therefore, provide suitable targets for therapeutic and biomarker discoveries (16).Proteomic tools have been proposed as a new platform for studying complex biological functions, which entail large numbers and networks of proteins (17). Moving beyond the imposing burden of providing lists of proteins identified in certain samples, the field of quantitative proteomics yields information that specifically recognizes the differences between samples and has emerged as a very important area of research in the study of cancer. These proteomics approaches have been extensively applied to cell secretome analyses for the elucidation of disease mechanisms, diagnoses, and new drug developments (16, 18–23). To comprehensively understand the roles of the secretion-related regulations in metastatic progression, the label-free quantitative proteomics approach was used to identify metastatic-associated proteins secreted by lung adenocarcinoma cells. Comparative secretome analysis was conducted in lung adenocarcinoma cells with differing levels of migration and invasiveness (CL1-0 versus CL1-5) (24). The CL1 cell lines have been used in previous metastasis research as novel protein targets associated with lung cancer metastasis discovery (25–28). The characterized protein, A1AT, was validated for its association and functions involved with lung adenocarcinoma metastasis by subjecting the cells to experimental metastasis assays in vitro and in vivo.     

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining demonstrated expression of β-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ∼95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises <1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8–10).Dental enamel formation is divided into secretory, transition, and maturation stages (11, 12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides (3, 13, 14). By the end of the secretory stage the enamel crystals are full-length and the enamel layer as a whole is as thick as it will ever be, but it has only about 14% of the mineral as it will have when the tooth erupts (15). Following the secretory stage there is then a transition during which the ameloblasts greatly reduce their secretion of enamel proteins (16) and convert to maturation ameloblasts (17). During the maturation stage, mineral is deposited exclusively on the sides of pre-existing enamel crystallites (18), which grow in width and thickness until further growth is prevented by contact with adjacent crystals (19, 20). During early maturation the percentage protein by weight drops from 30 to 2% (5), and half of the total enamel mineral is deposited. The final 30–35% of mineral is deposited in the absence of significant protein and allows the crystals to grow firmly against one another and to mechanically interlock (15).The major secretory stage enamel proteins are amelogenin (21, 22), ameloblastin (23–25), and enamelin (26, 27). These proteins function specifically during enamel formation, and the disease phenotypes exhibited by mice lacking these genes are confined to the developing teeth and include enamel agenesis (28–30). These genes are often deleted or are reduced to pseudogenes in vertebrates such as birds or baleen whales that evolved alternatives to developing teeth (31, 32). Although the enamel extracellular matrix proteins are critical for growing enamel crystals, they are not part of the final enamel product. Prior to tooth eruption, enamel proteins are digested by proteases and reabsorbed by ameloblasts. Two extracellular matrix proteases are involved in the cleavage of enamel proteins: matrix metalloproteinase 20 (Mmp-20)² (33) and kallikrein 4 (Klk4) (34).Mmp-20 is secreted along with amelogenin, ameloblastin, and enamelin by secretory stage ameloblasts (35–37). Mmp-20 activity can account for the range of cleavages observed in secretory stage enamel proteins (38) and appears to be the only protease secreted by ameloblasts during the secretory stage. Mmp20-null mice have enamel that is thinner and softer than normal, lacks enamel rod organization, and tends to chip off the crown surface (39, 40). Like the other secretory stage enamel proteins, Mmp20 expression appears to be restricted to developing teeth (41), as is the diseased phenotype when the human gene is defective (42–44).Klk4 is a serine protease that is secreted by transition and maturation stage ameloblasts but is not expressed by secretory stage ameloblasts (45, 46). Klk4 might also be expressed by odontoblasts, the cells that form dentin (47). Klk4 has broad substrate specificity (48, 49) and is capable of activating other proteases (50–52) and protease activated receptors (53, 54). Unlike most proteins secreted by ameloblasts, Klk4 is expressed in other tissues, most notably the prostate (55) and endometrium (56). Much attention has been focused on the potential role of Klk4 in cancers. Klk4 is increased in breast cancer stromal cells (57), in prostate cancer cells (58–61), and ovarian cancer cells (62–65). Despite this focus on the potential role of Klk4 in tumors, very little is known about the normal expression and function of Klk4 in nondental tissues. A loss of function mutation in both human KLK4 alleles caused a hypomaturation enamel phenotype in the absence of any observable defects elsewhere in the body (66).To gain insights into the role of Klk4 in normal dental enamel formation, and to better characterize the normal temporal and spatial patterns of Klk4 expression, we have used gene targeting to knock out normal Klk4 expression, while replacing the Klk4 code with lacZ, the bacterial gene encoding β-galactosidase reporter in mice. We demonstrate that Klk4 is not expressed by secretory stage ameloblasts, but is specifically expressed by ameloblasts later in enamel formation and is necessary for the proper removal of enamel proteins, the final thickening of enamel crystals, and ultimately, for hardening of the enamel layer.     

Outside the Unusual Cell Wall of the Hyperthermophilic Archaeon Aeropyrum pernix K1

In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.The archaea are a unique group of organisms that share properties with both the eukarya and bacteria. For a long time, archaeal life was considered to be limited to extreme environments such as high temperature, alkaline and acidic hot springs, anaerobic sediments, and highly saline environments. In the last decade, by the use of the archaeal 16 S rRNA gene as a molecular marker in microbial surveys (1), numerous mesophilic species have also been detected (2). Archaea have been found frequently and sometimes closely associated with bacterial and eukaryotic host cells, including humans. One of the most intriguing aspects of archaea is their unusual barrier between the inner cell material and the cellular environment, i.e. their cell membrane. Biosynthesis of archaeal cell wall has been a subject of interest for a long time. Most of the archaeal species characterized so far have a single chemically distinct cell membrane, which differs considerably from their eukaryotic and bacterial counterparts (3). The ether-type polar lipid surface is covered by a surface layer (S-layer)¹ composed of glycoproteins crystallized in regular two-dimensional lattices with hexagonal or tetragonal symmetry (4, 5). The structural characterization of the S-layer (6, 7) and S-layer-embedded archaeal cellular appendices such as flagella (8), pili, and hami (7, 9) associated with a diverse arsenal of cellular functions like motility, cell-cell communication, signaling, adherence, and nutrient uptake, has been the subject of an increasingly significant number of studies. Protein secretion mechanisms through this unusual cell membrane have been mainly addressed by way of comparative genomics studies (10–12) and by genomic identification and characterization of signal peptidases (13, 14). Archaeal extracellular and cell membrane proteins have been predicted because of the presence of a tripartite N-terminal signal motif essential for protein secretion and subsequently cleaved by signal peptidases from the protein (11, 14, 15). In archaea three different signal peptidases have been identified and characterized so far (13): signal peptidase I is responsible for the cleavage of secretory signal peptides from the majority of secreted proteins, class III signal peptidase is responsible for processing signal peptides from preflagellins and some sugar-binding proteins (11), and signal peptide peptidase is responsible for the hydrolysis of signal peptides following protein secretion. No signal peptidase II homolog in archaea has been described to date. Four distinct pathways have been proposed for archaeal protein export: the main “Sec” system, the twin arginine translocation or “Tat” pathway (12), the ATP-binding cassette (ABC) transport system (16), and the type IV prepilin-like pathway (11). Moreover, proteins without signal peptides could also be secreted by using nonspecific and/or currently unknown mechanisms. Despite the similarities in protein translocation mechanisms between the three domains of life, genome analyses also shed light on unique archaeal characteristics, suggesting that our current knowledge regarding secreted proteins and secretion mechanisms in archaea remains limited (10). It is apparent that the lack of experimental data at the proteome level has become the bottleneck for the further understanding of the existence of novel secretion mechanisms in archaea (15).To date, the genome sequences of eight hyperthermophiles, including the crenarchaeon Aeropyrum pernix K1, have been determined. A. pernix K1, isolated from a coastal solfataric thermal vent on the Kodakara-Jima Island in Japan (17), is the first reported obligate aerobic and neutrophilic hyperthermophilic archaeon with an optimal growth temperature between 90 and 95 °C. The spherical shaped cells of A. pernix are ∼1 μm in diameter, lack a rigid cell wall, and are covered by an S-layer with hexagonal symmetry. A. pernix, like other extreme thermophiles and acidophiles, possesses a particularly thick cell membrane that acts as a protective barrier, conferring to it the ability to function in the extreme environment in which it thrives. The lipids of A. pernix are different from those of anaerobic sulfur-dependent hyperthermophiles; they lack tetraether lipids and the direct linkage of inositol and sugar moieties (18). A. pernix K1 contains a 1.6-Mbp chromosome that has been sequenced; it comprises 1700 annotated genes. By using different proteomics approaches, the proteome of A. pernix K1 has recently been analyzed, leading to the identification of 704 proteins (41% of total ORFs) (19). In this work we performed proteomics analysis of the cell surface and extracellular protein fractions purified from A. pernix K1 to define proteins targeted to the cell secretome. We also analyzed the complete predicted proteome of A. pernix K1 by in silico signal peptide and cellular localization prediction tools and compared the experimentally obtained data set with the predicted secretome.     

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.Gram-negative bacteria have evolved several specialized protein secretion systems to secrete a wide variety of substrate proteins into the extracellular milieu or to inject them into other, often eukaryotic, cells (1). Secreted proteins and their associated secretion systems are very important in bacterial virulence and interactions with other organisms (2). One of the most recent discoveries in this field is the Type VI secretion system (T6SS),¹ which occurs widely across bacterial species (3, 4) and can target proteins to both bacterial and eukaryotic cells (5). The significance of the T6SS is becoming increasingly apparent. It has been implicated in virulence, commensalism, and symbiosis with eukaryotes (5, 6). Additionally, in many bacteria, the T6SS is now implicated in antibacterial activity. T6SS-mediated antibacterial killing appears to be important for competition between bacterial species, for example within the resident microflora of a eukaryotic host (5, 7).Secretion by the T6SS relies on 13 conserved core components which are predicted to form a large machinery associated with the cell envelope, including membrane-bound and bacteriophage tail-like subassemblies (8, 9). The membrane bound subassembly consists of inner membrane proteins (TssLM) and an outer membrane lipoprotein (TssJ) and is anchored to the cell wall. The phage tail-like assembly consists of several proteins that show structural homology with T4 phage tail proteins or are organized in similar structures (10). Hcp (TssD) proteins form hexameric rings and are thought to stack into tube-like structures (11, 12). This Hcp tube is believed to be capped by a trimer of VgrG (TssI) proteins, which share structural homology with the needle of the T4 phage tail (10, 13). In addition, VipA (TssB) and VipB (TssC) form a large tubular structure highly reminiscent of the T4 phage tail sheath (14, 15). Such similarities have led to the idea that the T6SS resembles an inverted contractile bacteriophage infection machinery and injects substrates via an Hcp/VgrG needle into other cells. Recent models propose that the VipA/B sheath surrounds the Hcp/VgrG needle and contraction of the VipA/B tube pushes the Hcp/VgrG needle out of the cell (16–18). It has been postulated that this mechanism can be triggered by close contact with other neighboring cells (19–21).Assembly, localization, and remodelling of VipA/B tubules in vivo depend on the AAA+ ATPase ClpV (TssH), another essential core component of the T6SS (14, 16, 17). ClpV also interacts with the accessory component Fha (TagH) (22, 23), which is found in a subset of T6SSs (4). The Fha protein has an N-terminal domain with a forkhead associated motif, which is predicted to bind phospho-threonine peptides (24). In Pseudomonas aeruginosa, Fha1 is phosphorylated by the Thr/Ser kinase PpkA (TagE) and dephosphorylated by the phosphatase PppA (TagG), and the phosphorylation state of Fha1 regulates the activity of the T6SS (22, 23). Phosphorylation of Fha in P. aeruginosa is also controlled by additional components, which act upstream of PpkA and form a regulatory cascade for T6SS activation (22, 25). Although homologs of PpkA and PppA have been identified in the T6SS gene clusters of certain other bacteria (3), the regulation of the T6SS by post-translational protein phosphorylation has not yet been experimentally investigated outside of Pseudomonas.To understand how the T6SS affects eukaryotic and bacterial cells, it is critical to identify substrate proteins secreted by the T6SS. The VgrG and Hcp proteins were the first identified T6SS substrates and appear to be generally secreted to the external milieu by all T6SSs (26). However, as mentioned above, Hcp and VgrG are core components of the T6SS machinery and therefore represent extracellular components of the secretion apparatus rather than genuine secreted effector proteins. Nonetheless, a limited number of VgrG homologs with extra functional effector domains at the C terminus have been identified or predicted, which account for some of the T6SS dependent effects seen against bacteria and eukaryotes. For example, the C-terminal domain of VgrG-1 from Vibrio cholerae shows actin crosslinking activity in eukaryotic cells (13, 27) and the C-terminal domain of V. cholerae VgrG-3 has bacterial cell wall hydrolase activity (28, 29).Recently, following much effort in the field, a small number of proteins secreted by the T6SS, but not structural components, have been experimentally identified. These proteins are regarded as true secreted substrates of the T6SS, with effector functions in target cells (29–35). For example, antibacterial T6SS-secreted effector proteins with peptidoglycan amidase (cell wall hydrolysis) function, the Type VI amidase effector (Tae) proteins, have been identified in Burkholderia thailandensis (32), P. aeruginosa (31), and Serratia marcescens (30). These Tae proteins play a role in T6SS-mediated antibacterial killing activity and genes encoding four families of Tae protein have been widely identified in other bacteria with T6SSs (32). T6SS-secreted effector proteins which are not peptidoglycan hydrolases have also been reported, including Tse2 secreted by P. aeruginosa, which acts in the bacterial cytoplasm (31), and the VasX and TseL proteins secreted by the V. cholerae T6SS, which are suggested to target membrane lipids (29, 34, 35). In the case of antibacterial T6SSs, the secreting bacterial cells are protected from their own T6SS effector proteins by specific immunity proteins (29–32, 35). However, given the large number of T6SSs in different bacterial species and their apparent ability to secrete multiple substrates, experimentally identified T6-secreted effector proteins still remain surprisingly scarce.Here we report the identification of multiple T6SS-secreted effector proteins in S. marcescens. S. marcescens is an opportunistic pathogen, for example causing ocular infections, nosocomial septicemia and pneumonia (36). Previously, we have identified a T6SS in S. marcescens Db10, which targets and efficiently kills other bacterial cells and plays a role in antibacterial competition (37). We have recently demonstrated that this T6SS secretes two antibacterial effectors, the Tae4 homologs Ssp1 and Ssp2, with cognate immunity proteins Rap1a and Rap2a (30).In this work, we report the analysis of the T6SS-dependent secretome of S. marcescens by label-free quantitation (LFQ) mass spectrometry and describe the identification and characterization of four novel T6SS-secreted effector proteins. These were confirmed as antibacterial toxins and specific immunity proteins were identified. Additionally, this global secretomic analysis, in combination with genetic and phosphoproteomic analyses, demonstrated that a post-translational phosphorylation system influences the ability of the S. marcescens T6SS to secrete effector proteins. Although this system uses homologs of the P. aeruginosa PpkA, PppA and Fha components, the circumstances and impact of Fha phosphorylation were shown to vary between organisms.     

Proteomic Analysis of the Palmitate-induced Myotube Secretome Reveals Involvement of the Annexin A1-Formyl Peptide Receptor 2 (FPR2) Pathway in Insulin Resistance

Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity.The obesity epidemic has been linked to the development of metabolic complications such as hyperlipidemia, insulin resistance, and hypertension (1, 2). Hyperlipidemia/dyslipidemia involves abnormally elevated levels of lipids and/or lipoproteins in the plasma (3, 4). Obese patients exhibit characteristics of hyperlipidemia/dyslipidemia, such as abnormal elevations in plasma free fatty acid, cholesterol, and triglyceride levels, as well as a reduction in high-density lipoprotein content (3–5). Elevated free fatty acid levels in the plasma of obese patients play an important role in the development of insulin resistance (6). Hence, lowering the free fatty acid level in plasma has been shown to restore insulin sensitivity in these patients (7). Palmitate (C16:0) is a saturated free fatty acid found in animal plasma. It has been reported that the concentration of plasma palmitate in obese patients is higher than in healthy individuals (6, 8). In molecular studies, palmitate has been found to induce inflammation and insulin resistance in skeletal muscle cells by promoting diacylglycerol accumulation, which in turn activates protein kinase C (PKC)-θ¹ and NF-κB, leading to the inhibition of insulin-stimulated Akt phosphorylation through insulin receptor substrate 1 (IRS1) (S307) phosphorylation and IL-6 secretion (9). Sortilin was recently identified as a mediator of palmitate-dependent insulin resistance, which regulates insulin-induced glucose transporter type 4 (GLUT4) trafficking (10). Therefore, palmitate is an important hyperlipidemic/dyslipidemic component that induces insulin resistance in skeletal muscle cells.Skeletal muscle is thought to function as a tissue that produces and releases cytokines called myokines (11). As part of its extracellular signaling pathway, skeletal muscle secretes myokines that participate in myogenesis, angiogenesis, and nutrient generation in response to factors such as metabolic disorders, including insulin resistance, and exercise (11–13). Some myokines, including IL-6, IL-8, IL-15, and fibroblast growth factor 21, and brain-derived neurotrophic factor (14), are induced by exercise. Although myokines are thought to play a critical role in the regulation of (patho)physiological processes, few studies have investigated the role of myokine in metabolism. Because skeletal muscle has a major role in the regulation of glucose metabolism, it is important to identify putative crucial regulators, secreted from skeletal muscle, that modulate glucose metabolism by acting as autocrine/paracrine mediators as well as endocrine mediators (15).Here, using an optimized secretomics approach, we performed a proteomic analysis of proteins in conditioned media from myotube cultures that were either untreated or treated with palmitate to induce insulin resistance (16, 17). Using a label-free quantitative analysis method, our aim was to characterize the skeletal muscle secretome and to identify skeletal muscle-derived proteins whose secretion is modulated by palmitate-induced insulin resistance. We found 36 putative secretory proteins modulated by palmitate-induced insulin resistance. The secretion of annexin A1 was down-regulated after palmitate treatment, and the annexin A1-formyl peptide receptor 2 (FPR2) pathway played a role in palmitate-induced insulin resistance in skeletal muscle by modulating the PKC-θ pathway.     

Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages

Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of unconventional protein secretion.The innate immune system is activated in response to microbial infection and tissue damage. Macrophages are the central players of the innate immunity and detect the presence of pathogen-associated molecular patterns (PAMPs)¹ and damage-associated molecular patterns (DAMPs) with their pattern recognition receptors. This recognition results in the activation of antimicrobial defense, inflammatory response, tissue regeneration, and recruitment of other inflammatory cells to the site of infection and/or tissue damage (1). Proper innate immune response is essential for the activation of the adaptive immune system. At present it is thought that the activation of innate immunity is most effective when both signals of microbial origin and damage are perceived at the same time (2, 3).Monosodium urate (MSU) is an endogenous DAMP that is crystallized from uric acid released by injured cells (4). Uric acid is a byproduct of purine degradation, and abnormally high levels of uric acid in serum, or hyperuricemia, is a hallmark of metabolic disorders where balance between intake of purines via food and excretion of uric acid is distorted. A well-known disease associated to hyperuricemia is gouty arthritis, in which deposits of MSU can be found in synovial fluid of peripheral joints, and MSU-induced inflammation is the initial trigger of symptoms (5). Hyperuricemia is also linked to other inflammatory diseases, like metabolic syndrome (6, 7), type 2 diabetes (8), and cardiovascular disease (9). MSU-induced inflammation is driven by the innate immune system. MSU engages antigen-presenting cells, macrophages, and dendritic cells. It is a potent adjuvant, initiating a robust adaptive immune response (4). Recently it has been shown that the adjuvant properties of alum are dependent on release of uric acid in vivo (10).It is unclear how cells detect the presence of MSU. It has been suggested that MSU activates intracellular signaling pathways in dendritic cells by directly engaging cellular membranes, particularly the cholesterol-rich components of the plasma membrane (11). Recently Uratsuji and coworkers showed that MSU activates inflammatory response in keratinocytes and monocytic THP-1 cells through membrane-associated P2Y6 (12). It is also well-documented that MSU activates the NLRP3 inflammasome in macrophages (13). The NLRP3 inflammasome is a multiprotein complex comprising of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), Apoptosis-associated speck-like protein containing a CARD (ASC) and cysteine protease Caspase-1. Activation of NLRP3 inflammasome results in the autocleavage of Caspase-1. The activated Caspase-1 then in turn cleaves pro-inflammatory cytokines IL-1β and IL-18 into their biologically active forms, which are then readily secreted (14–17). However, the signaling pathways that are involved in MSU-induced NLRP3 inflammasome activation have remained only partially characterized.Macrophages respond to activating stimuli by producing inflammatory mediators that are delivered to neighboring cells through multiple protein secretion pathways including both conventional and unconventional protein secretion (18). Conventionally secreted proteins contain an N-terminal signal peptide, which directs their transport to the plasma membrane through the well-characterized endoplasmic reticulum (ER)-Golgi pathway. In contrast, mediators and regulators of unconventional protein secretion pathways are less well understood. At present, different proteomic techniques allow for in-depth analysis of the secretome, the global pattern of secreted proteins. Secretome analysis is important in revealing complex cellular processes that require communication and signaling between the cells, and it has recently been applied to analyze the signaling pathways related to cell differentiation (19, 20), cancer (21, 22), and immune responses (23–25). In the present work we have characterized the secretome of human primary macrophages that have been activated simultaneously by microbial signal lipopolysaccharide (LPS) and endogenous danger signal MSU to get a global view of macrophage response to combined PAMP and DAMP stimulation.