Phosphatidylinositol transfer proteins: emerging roles in cell proliferation, cell death and survival

The actual cellular functions of the highly homologous small isoforms of the phosphatidylinositol transfer proteins, PI-TPalpha and PI-TPbeta have been studied using many different experimental conditions varying from in vitro experiments with purified proteins and lipid vesicles to investigations in animals. In this review, the very diverse data of these investigations have been collected and joined to propose a model for the cellular functions of PI-TPalpha and PI-TPbeta. The model is based on the suggested roles of PI-TPalpha and PI-TPbeta in various lipid-mediated cellular signaling pathways and leads to the conclusion that both proteins have a regulating function in pathways involved in the proliferation, apoptosis as well as survival of cells.     

The viral control of cellular acetylation signaling

It is becoming clear that the post-translational modification of histone and non-histone proteins by acetylation is part of an important cellular signaling process controlling a wide variety of functions in both the nucleus and the cytoplasm. Recent investigations designate this signaling pathway as one of the primary targets of viral proteins after infection. Indeed, specific viral proteins have acquired the capacity to interact with cellular acetyltransferases (HATs) and deacetylases (HDACs) and consequently to disrupt normal acetylation signaling pathways, thereby affecting viral and cellular gene expression. Here we review the targeting of cellular HATs and HDACs by viral proteins and highlight different strategies adopted by viruses to control cellular acetylation signaling and to accomplish their life cycle.     

Post-translational modifications of host proteins by Legionella pneumophila: a sophisticated survival strategy

Eukaryotic proteins are tightly regulated by post-translational modifications, leading to a very subtle degree of regulation in time and space. Pathogen-mediated post-translational modifications are key strategies to modulate host factors by targeting central signaling pathways in the host cell. Legionella pneumophila, an intracellular pathogen that coevolved with protozoan hosts, encodes a large arsenal of secreted effectors conferring the ability to evade host cellular defenses and to manipulate them to promote invasion and intracellular replication. Conservation of many signaling pathways of protozoa in human macrophages confers the ability of L. pneumophila to infect humans, causing a severe pneumonia called legionnaires' disease. Most of the secreted proteins are delivered by the Dot/Icm type IV secretion system and several of these have been shown to act on different cellular pathways critical for infection. Moreover, multiple effectors target a single host function to orchestrate bacterial survival. In this review, we focus on those effectors in the repertoire of L. pneumophila proteins that target key cellular pathways by specific post-translational modifications.     

Computational exploration of the activated pathways associated with DNA damage response in breast cancer

Molecular signaling events regulate cellular activity. Cancer stimulating signals trigger cellular responses that evade the regulatory control of cell development. To understand the mechanism of signaling regulation in cancer, it is necessary to identify the activated pathways in cancer. We have developed RepairPATH, a computational algorithm that explores the activated signaling pathways in cancer. The RepairPATH integrates RepairNET, an assembled protein interaction network associated with DNA damage response, with the gene expression profiles derived from the microarray data. Based on the observation that cofunctional proteins often exhibit correlated gene expression profiles, it identifies the activated signaling pathways in cancer by systematically searching the RepairNET for proteins with significantly correlated gene expression profiles. Analyzing the gene expression profiles of breast cancer, we found distinct similarities and differences in the activated signaling pathways between the samples from the patients who developed metastases and the samples from the patients who were disease free within 5 years. The cellular pathways associated with the various DNA repair mechanisms and the cell-cycle checkpoint controls are found to be activated in both sample groups. One of the most intriguing findings is that the pathways associated with different cellular processes are functionally coordinated through BRCA1 in the disease-free sample group, whereas such functional coordination is absent in the samples from patients who developed metastases. Our analysis revealed the potential cellular pathways that regulate the signaling events in breast cancer.     

Protein fatty acylation: a novel mechanism for association of proteins with membranes and its role in transmembrane regulatory pathways

A wide range of proteins of cellular and viral origin have been shown to be modified covalently by long-chain fatty acids. Recent studies have revealed at least two distinct types of protein fatty acylation which involve different fatty acyltransferases. The abundant fatty acid, palmitate, is incorporated post-translationally through a thiol ester linkage into a variety of cell surface glycoproteins and non-glycosylated intracellular proteins. In contrast, the rare fatty acid, myristate, is incorporated co-translationally through an amide linkage into numerous intracellular proteins. Identification of proteins that contain covalent fatty acids has revealed that this modification is common to a broad array of proteins that play important roles in transmembrane regulatory pathways. For many of these proteins, the fatty acid moiety appears to play an important role in directing the polypeptide to the appropriate membrane and in mediating protein-protein interactions within the membrane. This review will summarize recent studies that define different pathways for protein fatty acylation and will consider the potential functions for this unique covalent modification of proteins.     

"Omic" approaches for unraveling signaling networks

Signaling pathways are crucial for cell differentiation and response to cellular environments. Recently, a large number of approaches for the global analysis of genes and proteins have been described. These have provided important new insights into the components of different pathways and the molecular and cellular responses of these pathways. This review covers genomic and proteomic (collectively referred to as "omic") approaches for the global analysis of cell signaling, including gene expression profiling and analysis, protein-protein interaction methods, protein microarrays, mass spectroscopy and gene-disruption and engineering approaches.     

Proteomic analysis of different temporal expression patterns induced by N-methyl-N'-nitro-N-nitrosoguanidine treatment

We have previously shown that N-methyl- N'-nitro- N-nitrosoguanidine (MNNG), a well-known DNA alkylating agent and carcinogen, can induce multiple cellular responses with dynamic characteristics, including such responses as nontargeted mutations (NTM) at undamaged bases in DNA, up-regulation of low fidelity DNA polymerases, clustering of epidermal growth factor receptor (EGFR) and interference with its downstream signaling pathway. A dose-related analysis also revealed that different concentrations of MNNG can trigger diverse proteome changes associated with different cytotoxic effects. To further understand the dynamic cellular responses and hazardous effects caused by environmental carcinogen, a proteomic time-course study of whole cellular proteins from human amniotic epithelial cells after MNNG treatment was performed. Analysis at three different time points (3, 12 and 24 h after exposure) revealed that the major changes were taking place around 3 and 12 h after exposure. Using MALDI-TOF MS coupled with a micro solid-phase extraction (SPE) device, 90% ( n = 70) differentially expressed proteins were identified. Functional assignment revealed that many important pathways were affected, including the protein biosynthesis pathway and Ran GTPase system. We also carried out a network analysis of these proteins and the data suggest a central role for some key regulators in different pathways.     

PSMA2 knockdown impacts expression of proteins involved in immune and cellular stress responses in human lung cells

Proteasome subunit alpha type-2 (PSMA2) is a critical component of the 20S proteasome, which is the core particle of the 26S proteasome complex and is involved in cellular protein quality control by recognizing and recycling defective proteins. PSMA2 expression dysregulation has been detected in different human diseases and viral infections. No study yet has reported PSMA2 knockdown (KD) effects on the cellular proteome. Methods: We used SOMAScan, an aptamer-based multiplexed technique, to measure >1300 human proteins to determine the impact of PSMA2 KD on A549 human lung epithelial cells. Results: PSMA2 KD resulted in significant dysregulation of 52 cellular proteins involved in different bio-functions, including cellular movement and development, cell death and survival, and cancer. The immune system and signal transduction were the most affected cellular functions. PSMA2 KD caused dysregulation of several signaling pathways involved in immune response, cytokine signaling, organismal growth and development, cellular stress and injury (including autophagy and unfolded protein response), and cancer responses. Conclusions: In summary, this study helps us better understand the importance of PSMA2 in different cellular functions, signaling pathways, and human diseases.     

High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells

It has been almost a decade since the last review appeared comparing and contrasting the influences that the different families of High Mobility Group proteins (HMGA, HMGB and HMGN) have on the various DNA repair pathways in mammalian cells. During that time considerable progress has been made in our understanding of how these non-histone proteins modulate the efficiency of DNA repair by all of the major cellular pathways: nucleotide excision repair, base excision repair, double-stand break repair and mismatch repair. Although there are often similar and over-lapping biological activities shared by all HMG proteins, members of each of the different families appear to have a somewhat ‘individualistic’ impact on various DNA repair pathways. This review will focus on what is currently known about the roles that different HMG proteins play in DNA repair processes and discuss possible future research areas in this rapidly evolving field.     

The 14-3-3 proteins in regulation of cellular metabolism

Thirty years ago, it was discovered that 14-3-3 proteins could activate enzymes involved in amino acid metabolism. In the following decades, 14-3-3s have been shown to be involved in many different signaling pathways that modulate cellular and whole body energy and nutrient homeostasis. Large scale screening for cellular binding partners of 14-3-3 has identified numerous proteins that participate in regulation of metabolic pathways, although only a minority of these targets have yet been subject to detailed studies. Because of the wide distribution of potential 14-3-3 targets and the resurging interest in metabolic pathway control in diseases like cancer, diabetes, obesity and cardiovascular disease, we review the role of 14-3-3 proteins in the regulation of core and specialized cellular metabolic functions. We cite illustrative examples of 14-3-3 action through their direct modulation of individual enzymes and through regulation of master switches in cellular pathways, such as insulin signaling, mTOR- and AMP dependent kinase signaling pathways, as well as regulation of autophagy. We further illustrate the quantitative impact of 14-3-3 association on signal response at the target protein level and we discuss implications of recent findings showing 14-3-3 protein membrane binding of target proteins.     

Poxvirus antagonism of innate immunity by Bcl-2 fold proteins

Poxviruses have evolved numerous mechanisms to evade host innate immunity. Sensory pathways that are activated by Toll-like and nucleotide receptors, as well as innate cell death pathways, are both targets of antagonism by viral proteins. Recent structural, biochemical and functional studies of poxvirus proteins have identified a family of α-helical proteins that adopt a Bcl-2 fold despite highly divergent polypeptide sequences from cellular proteins that regulate apoptosis. These newly identified proteins have assumed new roles in antagonism of NF-κB and interferon signaling pathways and interfere with the release of pro-inflammatory cytokines. Structures of isolated viral proteins and their complexes with cellular targets provide insight into the diverse ways that the Bcl-2 scaffold can be exploited for antagonism of host immunity.     

Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis

The production of ROS is an inevitable consequence of metabolism. However, high levels of ROS within a cell can be lethal and so the cell has a number of defences against oxidative cell stress. Occasionally the cell's antioxidant mechanisms fail and oxidative stress occurs. High levels of ROS within a cell have a number of direct and indirect consequences on cell signalling pathways and may result in apoptosis or necrosis. Although some of the indirect effects of ROS are well known, limitations in technology mean that the direct effects of the cell's redox environment upon proteins are less understood. Recent work by a number of groups has demonstrated that ROS can directly modify signalling proteins through different modifications, for example by nitrosylation, carbonylation, di-sulphide bond formation and glutathionylation. These modifications modulate a protein's activity and several recent papers have demonstrated their importance in cell signalling events, especially those involved in cell death/survival. Redox modification of proteins allows for further regulation of cell signalling pathways in response to the cellular environment. Understanding them may be critical for us to modulate cell pathways for our own means, such as in cytotoxic drug treatments of cancer cells. Protein modifications mediated by oxidative stress can modulate apoptosis, either through specific protein modifications resulting in regulation of signalling pathways, or through a general increase in oxidised proteins resulting in reduced cellular function. This review discusses direct oxidative protein modifications and their effects on apoptosis.     

Immunocell-array for molecular dissection of multiple signaling pathways in mammalian cells

The knowledge of signaling pathways that are triggered by physiological and pathological conditions or drug treatment is essential for the comprehension of the biological events that regulate cellular responses. Recently novel platforms based on "reverse-phase protein arrays" have proven to be useful in the study of different pathways, but they still lack the possibility to detect events in the complexity of a cellular context. We developed an "immunocell-array" of cells on chip where, upon cell plating, growing, drug treatment, and fixation, by spotting specific antibodies we can detect the localization and state of hundreds of proteins involved in specific signaling pathways. By applying this technology to mammalian cells we analyzed signaling proteins involved in the response to DNA damage and identified a chromatin remodeling pathway following bleomycin treatment. We propose our technology as a new tool for the array-based multiplexed analysis of signaling pathways in drug response screening, for the proteomics of profiling patient cells, and ultimately for the high throughput screening of antibodies for immunofluorescence applications.     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.