Proteomics: posttranslational modifications, immune responses and current analytical tools

The publication of the human genome sequence enables most of the still unknown protein sequences to be added to the current databases. A sequence alone does not, however, give information about the possible expression level of the corresponding protein, neither does it inform about the possible posttranslational modifications, like phosphorylation, glycosylation or changes in individual amino acids. Thus, the human proteome project, a large scale analysis of the functions of gene products, will have an enormous impact on our understanding of the biochemistry of proteins, processes and pathways they are involved in. The diversity in proteins is considerably expanded by various posttranslational modifications. These also pose problems to the investigators, but their careful analysis often pays back because they can reveal important properties in proteins or peptides--like an increased antigenicity leading to (auto)immune responses or an active form of a signaling protein. Immune tolerance usually exists towards self-proteins, but in specific cases it may be broken by posttranslational modifications in the proteins. Novel mass spectrometric, affinity and display techniques offer valuable tools for the large-scale analysis of proteomes. In the present paper we discuss their use for the detection of posttranslational modifications, functional interactions and possible disease-associated abnormalities in proteins.     

Involvement of nuclear domains in the cellular response to DNA damage

All cells of human organism are continuously damaged, and a damage of the genetic material can be especially dangerous. The reaction of the cell to DNA damage is a complex process, which includes damage signaling, repair, apoptosis or cell death. It is connected with serious changes in the cell nucleus, which are caused by posttranslational modifications and dynamic relocalizations of proteins as well as alterations in the expression of many genes. These changes are not limited to the sites of DNA damage, but involve whole cell nucleus, including its domains: PML bodies, nucleolus and Cajal bodies.     

The roles of FOX proteins in virus-associated cancers

Forkhead box (FOX) proteins play a crucial role in regulating the expression of genes involved in multiple biological processes, such as metabolism, development, differentiation, proliferation, apoptosis, migration, invasion, and longevity. Deregulation of FOX proteins is commonly associated with cancer initiation, progression, and chemotherapeutic drug resistance in many human tumors. FOX proteins deregulate through genetic events and the perturbation of posttranslational modification. The purpose of the present review is to describe the deregulation of FOX proteins by oncoviruses. Oncoviruses utilize various mechanisms to deregulate FOX proteins, including alterations in posttranslational modifications, cellular localization independently of posttranslational modifications, virus-encoded miRNAs, activation or suppression of a series of cell signaling pathways. This deregulation can affect proliferation, metastasis, chemotherapy resistance, and immunosuppression in virus-induced cancers and help to chronic viral infection, development of gluconeogenic responses, and inflammation. Since the PI3K/Akt/mTOR signaling pathway is the upstream FOXO, suppressing it can cause FOXO function to return, and this can be one of the reasons for patients to recover from the infection of the viruses used to treat these inhibitors. Hence, FOX proteins could serve as prognosis markers and target therapy specifically in cancers caused by oncoviruses.     

Brain Actin Synthesized In Vitro Undergoes Two Different and Sequential Posttranslational Modifications

Abstract: We have studied the posttranslational processing of actin molecules synthesized in a cell-free system. The results of these experiments indicate that during the in vivo synthesis of the actins from rat brain the primary translational products undergo two different and sequential posttranslational modifications. These modifications are accompanied by slight changes in the isoelectric points of the proteins and can be detected by isoelectric focusing analysis. The same posttranslational modifications can be detected during the in vitro synthesis of chick embryo skeletal muscle actin. The evidence presented suggest that the first posttranslational modification may correspond to the methylation of a histidine residue, and the second modification most likely corresponds to the acetylation of the NH₂-terminal amino acid residues of actin molecules.     

Posttranslational modification of MDM2

The functions of the MDM2 protein, in particular its E3 ubiquitin ligase activity and its ability to interact with a number of cellular proteins intimately involved in growth regulation, are modulated by sumoylation and multisite phosphorylation. These posttranslational mechanisms not only regulate the intrinsic activity of MDM2 in response to cellular stresses, but also govern its subcellular localization, differentiate between MDM2-mediated ubiquitination of p53 and autoubiquitination, integrate the stress response with mechanisms that mediate cell survival, and modulate the interaction of MDM2 with cellular and viral proteins. In this review, we summarize our current knowledge of the role of posttranslational modifications of MDM2 and their functional relevance.     

Regulation of the water channel aquaporin-2 by posttranslational modification

The cellular functions of many eukaryotic membrane proteins, including the vasopressin-regulated water channel aquaporin-2 (AQP2), are regulated by posttranslational modifications. In this article, we discuss the experimental discoveries that have advanced our understanding of how posttranslational modifications affect AQP2 function, especially as they relate to the role of AQP2 in the kidney. We review the most recent data demonstrating that glycosylation and, in particular, phosphorylation and ubiquitination are mechanisms that regulate AQP2 activity, subcellular sorting and distribution, degradation, and protein interactions. From a clinical perspective, posttranslational modification resulting in protein misrouting or degradation may explain certain forms of nephrogenic diabetes insipidus. In addition to providing major insight into the function and dynamics of renal AQP2 regulation, the analysis of AQP2 posttranslational modification may provide general clues as to the role of posttranslational modification for regulation of other membrane proteins.     

Peroxynitrite transforms nerve growth factor into an apoptotic factor for motor neurons

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.     

VDAC proteomics: Post-translation modifications

Voltage-dependent anion channels are abundant mitochondrial outer membrane proteins expressed in three isoforms, VDAC1-3, and are considered as "mitochondrial gatekeepers". Most tissues express all three isoforms. The functions of VDACs are several-fold, ranging from metabolite and energy exchange to apoptosis. Some of these functions depend on or are affected by interaction with other proteins in the cytosol and intermembrane space. Furthermore, the function of VDACs, as well as their interaction with other proteins, is affected by posttranslational modification, mainly phosphorylation. This review summarizes recent findings on posttranslational modification of VDACs and discusses the physiological outcome of these modifications. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.     

同源结构域相互作用蛋白激酶2(HIPK2)研究进展

同源结构域相互作用蛋白激酶2(HIPK2)是一种定位于细胞核内的丝氨酸/苏氨酸蛋白激酶。HIPK2可以诱导和抑制转录,亦能调节细胞分化、增殖及凋亡。HIPK2经过泛素化、小泛素样修饰物(SUMO)化、乙酰化、磷酸化等翻译后修饰来发挥其生物功能。研究表明,HIPK2磷酸化p53、甲基化CpG结合蛋白2(methyl CpG binding protein 2,MeCP2)及早幼粒细胞性白血病蛋白(promyelocytic leukemia protein,PML)促进细胞凋亡。本文较为详细的阐述了有关HIPK2的研究进展,特别是近5年的研究成果。     

Protein prenyltransferases: anchor size,pseudogenes and parasites

Lipid modification of eukaryotic proteins by protein prenyltransferases is required for critical signaling pathways, cell cycle progression, cytoskeleton remodeling, induction of apoptosis and vesicular trafficking. This review analyzes the influence of distinct states of sequential posttranslational processing that can be obtained after single or double prenylation, reversible palmitoylation, proteolytic cleavage of the C-terminus and possible reversible carboxymethylation. This series of modifications, as well as the exact length of the prenyl anchor, are determinants in protein-membrane and specific protein-protein interactions of protein prenyltransferase substrates. Furthermore, the occurrence and distribution of pseudogenes of protein prenyltransferase subunits are discussed. Besides being developed as anti-cancer agents, prenyltransferase inhibitors are effective against an increasing number of parasitic diseases. Extensive screens for protein prenyltransferases in genomic data of fungal and protozoan pathogens unveil a series of new pharmacologic targets for prenyltransferase inhibition, including the parasites Brugia malayi, Onchocerca volvulus, Aspergillus nidulans, Pneumocystis carinii, Entamoeba histolytica, Strongyloides stercoralis, Trichinella spiralis and Cryptosporidium parvum.     

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.     

Regulation of BAD protein by PKA, PKCdelta and phosphatases in adult rat cardiac myocytes subjected to oxidative stress

H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases.     

Bcl-2 family members: integrators of survival and death signals in physiology and pathology [corrected

The members of the Bcl-2 family of proteins are crucial regulators of apoptosis. In order to determine cell fate, these proteins must be targeted to distinct intracellular membranes, including the mitochondrial outer membrane (MOM), the membrane of the endoplasmic reticulum (ER) and its associated nuclear envelope. The targeting sequences and mechanisms that mediate the specificity of these proteins for a particular cellular membrane remain poorly defined. Several Bcl-2 family members have been reported to be tail-anchored via their predicted hydrophobic COOH-terminal transmembrane domains (TMDs). Tail-anchoring imposes a posttranslational mechanism of membrane insertion on the already folded protein, suggesting that the transient binding of cytosolic chaperone proteins to the hydrophobic TMD may be an important regulatory event in the targeting process. The TMD of certain family members is initially concealed and only becomes available for targeting and membrane insertion in response to apoptotic stimuli. These proteins either undergo a conformational change, posttranslational modification or a combination of these events enabling them to translocate to sites at which they are functional. Some Bcl-2 family members lack a TMD, but nevertheless localize to the MOM or the ER membrane during apoptosis where they execute their functions. In this review, we will focus on the intracellular targeting of Bcl-2 family members and the mechanisms by which they translocate to their sites of action. Furthermore, we will discuss the posttranslational modifications which regulate these events.     

Tools for phospho- and glycoproteomics of plasma membranes

Analysis of plasma membrane proteins and their posttranslational modifications is considered as important for identification of disease markers and targets for drug treatment. Due to their insolubility in water, studying of plasma membrane proteins using mass spectrometry has been difficult for a long time. Recent technological developments in sample preparation together with important improvements in mass spectrometric analysis have facilitated analysis of these proteins and their posttranslational modifications. Now, large scale proteomic analyses allow identification of thousands of membrane proteins from minute amounts of sample. Optimized protocols for affinity enrichment of phosphorylated and glycosylated peptides have set new dimensions in the depth of characterization of these posttranslational modifications of plasma membrane proteins. Here, I summarize recent advances in proteomic technology for the characterization of the cell surface proteins and their modifications. In the focus are approaches allowing large scale mapping rather than analytical methods suitable for studying individual proteins or non-complex mixtures.