The cell aggregating propensity of probiotic actinobacterial isolates: isolation and characterization of the aggregation inducing peptide pheromone

The auto-aggregating ability of a probiotic is a prerequisite for colonization and protection of the gastrointestinal tract, whereas co-aggregation provides a close interaction with pathogenic bacteria. Peptide pheromone mediated signaling has been studied in several systems. However, it has not yet been explored in prokaryotes, especially actinobacteria. Hence, in the present study, the diffusible aggregation promoting factor was purified from the culture supernatant of a potent actinobacterial probiont and characterized using 20 different actinobacterial cultures isolated from the gut region of chicken and goat. The results showed that the pheromone-like compound induces the aggregation propensity of treated isolates. The factor was found to be a heat stable, acidic pH resistant, low molecular weight peptide which enhances the biofilm forming ability of other actinobacterial isolates. The aggregation promoting factor represents a bacterial sex factor (pheromone) and its characterization confirms its usage in the probiotic formulation     

Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons

Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.     

Environmental Stress Affects the Activity of Metabolic and Growth Factor Signaling Networks and Induces Autophagy Markers in MCF7 Breast Cancer Cells

Phosphoproteomic techniques are contributing to our understanding of how signaling pathways interact and regulate biological processes. This technology is also being used to characterize how signaling networks are remodeled during disease progression and to identify biomarkers of signaling pathway activity and of responses to cancer therapy. A potential caveat in these studies is that phosphorylation is a very dynamic modification that can substantially change during the course of an experiment or the retrieval and processing of cellular samples. Here, we investigated how exposure of cells to ambient conditions modulates phosphorylation and signaling pathway activity in the MCF7 breast cancer cell line. About 1.5% of 3,500 sites measured showed a significant change in phosphorylation extent upon exposure of cells to ambient conditions for 15 min. The effects of this perturbation in modifying phosphorylation patterns did not involve random changes due to stochastic activation of kinases and phosphatases. Instead, exposure of cells to ambient conditions elicited an environmental stress reaction that involved a coordinated response to a metabolic stress situation, which included: (1) the activation of AMPK; (2) the inhibition of PI3K, AKT, and ERK; (3) an increase in markers of protein synthesis inhibition at the level of translation elongation; and (4) an increase in autophagy markers. We also observed that maintaining cells in ice modified but did not completely abolish this metabolic stress response. In summary, exposure of cells to ambient conditions affects the activity of signaling networks previously implicated in metabolic and growth factor signaling. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000472.Phosphorylation is a posttranslational modification involving the addition of phosphate groups to serine, threonine and tyrosine residues on target proteins. This modification, regulated by kinases and phosphatases that phosphorylate and dephosphorylate these amino acids respectively, controls many aspects of protein biochemistry including stability, localization, ability to interact with other molecules and enzymatic activity (1, 2). In addition to playing a pivotal role in regulating most biological processes, alterations in biochemical pathways regulated by protein phosphorylation contribute to the pathophysiology of various diseases including cancer, diabetes and neurodegeneration (2–6).In recent years the development of MS techniques has allowed the study of protein phosphorylation on an untargeted and global scale. As a consequence, signaling processes can now be studied with unprecedented depth and coverage (7–10). Phosphoproteomics has also been applied to investigate how signaling networks are modulated during disease progression and for the identification of biomarkers that classify patients according to prognosis or treatment response (11–15). A potential caveat in the interpretation of such experiments is that protein phosphorylation is a dynamic modification that can be affected by variables difficult to control including cell confluence, circadian rhythms, shear stress and other types of environmental stresses including exposure to ambient conditions (16–22). Thus, during the course of an experiment variations or delays in sample retrieval and processing can potentially alter the quantitative characteristics of the phosphoproteome (17, 18, 22). Similar problems could in principle occur in a clinical environment where several hours may elapse from patient sample collection to processing or preservation (16, 17, 23). Delays because of ethical and practical considerations may also affect collection and preservation of post-mortem samples (24, 25). As a consequence, it can in principle be introduced variability and artifacts that may potentially confound the interpretation of data obtained from large-scale as well as targeted phosphoproteomics experiments (16).To our knowledge, there are no reports that systematically evaluate, in an untargeted manner, how exposure to environmental stress modulates the phosphoproteome of human cells in culture. Here, we used the MCF7 breast cancer cell line to investigate how ambient conditions alter phosphorylation and to evaluate signaling pathways that may be modulated by environmental stress. We found several phosphorylation events that increased or decreased after 15 min exposure of cells to ambient conditions at room temperature (RT)¹. We then studied whether these changes in phosphorylation were a random effect due to stochastic inactivation of kinases and phosphatases or whether these were the consequence of actual responses involving specific signaling pathways. Our data indicate that the phosphorylations regulated by environmental conditions at RT are the initial steps of a complex adaptive response to a metabolic stress. Data supporting these conclusions include the observation that ambient conditions at RT activated catabolic pathways regulated by AMPK and GSK3β and inactivated anabolic pathways involving the AKT, ERK and mTOR signaling nodes. When we compared the responses to ambient conditions at RT or on ice, we found that maintaining cells on ice induced a different adaptive response rather than an attenuated one. We also found that the adaptation response to ambient conditions at RT triggered a functional biological process that involved the initiation of macroautophagy (hereafter referred as autophagy) and the activation of a pathway known to inhibit protein synthesis at the level of translation elongation. Thus our study also defines experimental conditions that can be used to study the mechanisms involved in the process of autophagy.     

Cross-species Proteomics Reveals Specific Modulation of Signaling in Cancer and Stromal Cells by Phosphoinositide 3-kinase (PI3K) Inhibitors

The tumor microenvironment plays key roles in cancer biology, but its impact on the regulation of signaling pathway activity in cancer cells has not been systemically investigated. We designed an analytical strategy that allows differential analysis of signaling between cancer and stromal cells present in tumor xenografts. We used this approach to investigate how in vivo growth conditions and PI3K inhibitors regulate pathway activities in both cancer and stromal cell populations. We found that, despite inducing more modest changes in protein expression, in vivo growing conditions extensively rewired protein kinase networks in cancer cells. As a result, different sets of phosphorylation sites were modulated by PI3K inhibitors in cancer cells growing in tumors relative to when these cells were in culture. The p110δ PI3K-selective compound CAL-101 (Idelalisib) did not inhibit markers of PI3K activity in cancer or stromal cells; however, unexpectedly, it induced phosphorylation on SQ motifs in both subpopulations of tumor cells in vivo but not in vitro. Thus, the interaction between cancer cells and the stroma modulated the ability of PI3K inhibitors to induce the activation of apoptosis in solid tumors. Our study provides proof-of-principle of a proteomics workflow for measuring signaling specifically in cancer and stromal cells and for investigating how cancer biochemistry is modulated in vivo.Solid tumors contain a heterogeneous population of cells. Transformed epithelial cells recruit different types of somatic cells to the tumor microenvironment where they influence varying aspects of cancer biology. The role of heterotypic communication between normal stromal cells and transformed cancer cells is well established (1, 2). Different somatic cell types, including fibroblasts, epithelial cells, and cells of the immune system—all of which are found in tumors—promote cancer cell development by means of gap-junction intercellular communication, direct cell-to-cell contacts, and by the release of growth factors, enzymes, and cytokines that act on neighboring malignant cells (3–6).The tumor microenvironment determines the ability of cancer cells to survive in specific organs and their ability to proliferate and metastasize (7–9). Growth factors released from tumor-associated stromal cells also influence how cancer cells respond to drug administration (10). Therefore, the advancement of targeted cancer therapies requires an understanding of how the tumor microenvironment modulates the biochemistry of transformed cancer cells. In addition, targeting the tumor stroma is emerging as an intriguing concept for the development of anti-cancer therapies (11). It is therefore important to investigate specific effects of compounds in clinical development on stromal cells in addition to those exerted toward malignant cancer cells (12).Here we investigated the effects that changes in growing conditions from a two-dimensional cell culture to an in vivo three-dimensional tumor environment had in modulating protein and phosphoprotein expression in human cancer cells. For this, we used mass spectrometry (MS) to specifically measure cancer and stromal proteomes and phosphoproteomes within mouse tumor xenografts.We also investigated the effects that the pharmacological inhibitors of PI3K, namely GDC-0941 or CAL-101, would have on the phosphoproteomes of stromal cells relative to cancer cells in solid tumors. GDC-0941 is an inhibitor with specificity for class I PI3Ks, whereas CAL-101 specificity is restricted to the p110δ isoform of PI3K (13, 14), which in untransformed tissues is mainly found in leukocytes (15). The PI3K signaling pathway is often deregulated in different cancer types (16), including colorectal cancer (17), and both compounds used in this study are in different stages of clinical development (18–20). PI3K signaling has also been implicated in mediating the effects that the microenvironment has on cancer cells (21).We found that in vivo growth conditions had profound effects on phosphoprotein expression, which was reflected on the phosphorylation sites modulated by PI3K inhibitors in vivo relative to in vitro and in their ability to induce apoptotic markers across these two cell culture conditions.     

eEF2K Activity Determines Synergy to Cotreatment of Cancer Cells With PI3K and MEK Inhibitors

PI3K-mammalian target of rapamycin and MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK) are the most frequently dysregulated signaling pathways in cancer. A problem that limits the success of therapies that target individual PI3K-MAPK members is that these pathways converge to regulate downstream functions and often compensate each other, leading to drug resistance and transient responses to therapy. In order to overcome resistance, therapies based on cotreatments with PI3K/AKT and MEK/MAPK inhibitors are now being investigated in clinical trials, but the mechanisms of sensitivity to cotreatment are not fully understood. Using LC-MS/MS-based phosphoproteomics, we found that eukaryotic elongation factor 2 kinase (eEF2K), a key convergence point downstream of MAPK and PI3K pathways, mediates synergism to cotreatment with trametinib plus pictilisib (which target MEK1/2 and PI3Kα/δ, respectively). Inhibition of eEF2K by siRNA or with a small molecule inhibitor reversed the antiproliferative effects of the cotreatment with PI3K plus MEK inhibitors in a cell model–specific manner. Systematic analysis in 12 acute myeloid leukemia cell lines revealed that eEF2K activity was increased in cells for which PI3K plus MEKi cotreatment is synergistic, while PKC potentially mediated resistance to such cotreatment. Together, our study uncovers eEF2K activity as a key mediator of responses to PI3Ki plus MEKi and as a potential biomarker to predict synergy to cotreatment in cancer cells.