泛素-蛋白酶体与药物应用

In eukaryotes, the ubiquitin-proteasome pathway degrades the majority of intracellular proteins tagged with polyubiquitin chains. It participates in regulation of key cellular activities, such as cell proliferation, cell differentiation, apoptosis, DNA repair, etc. through the degradation of malformed or misfolded proteins. Dysfunctions of the ubiquitin-proteasome pathway have been linked to many diseases, including cancer and neurodegeneration, etc. The commercially available proteasome inhibitors have been successfully used to treat multiple myeloma and mantle cell lymphoma. In addition, novel inhibitors against other components of the ubiquitin-proteasome pathway, such as those enzymes that drive ubiquitination and deubiquitination in preclinical testing or clinical trials, exhibit promising therapeutic effects in vivo. This paper briefly introduces the ubiquitin-proteasome pathway related drug discovery progress.     

Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes

Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase and caspase-3 by nitric oxide

The regulation of enzyme activity function is a major factor in the cellular response to a changing environment. One mechanism of enzyme activity regulation includes post-translational protein thiol modification by nitric oxide (NO) or its redox species. Major routs used by NO to modify cysteine residues of proteins include S-nitrosation, oxidation, mixed disulfide formation with glutathione, and the covalent attachment of nucleotide cofactors, i.e NAD(+)/NADH. Critical thiol centers serve as recognition sites for NO, thus channeling the NO signal through post-translational modifications and oxidation into cellular functions. Here, we summarize current knowledge on active site thiol modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and caspase-3 by nitric oxide. Although very different in their cellular function, both enzymes contain highly reactive cysteines which represent sensitive targets for NO. Our studies are supportive of a potential role of S-nitrosation and mixed disulfide formation as a general signaling mechanism that allows sensing of nitrosative stress. At the same time, modification of GAPDH and caspase-3 by NO show the diversity of mechanisms (S-nitrosation versus oxidations) that we are confronted with as a result of NO delivery, especially comparing in vitro studies with cellular systems. In the future it will be challenging to dissect how nitrosative and oxidative signaling mechanisms overlap and how intracellular communication systems allow their activation in a selective way.     

Protein Prenylation: An (Almost) Comprehensive Overview on Discovery History, Enzymology, and Significance in Physiology and Disease

Summary. Since 1979, when prenylation has been first discovered as chemical oddity of a yeast mating factor, the two forms of this posttranslational modification of proteins (farnesylation and geranylgeranylation) have been found as wide spread among proteins from Eukarya and their viruses. This review attempts to summarize as comprehensively as possible the enzymological processes of prenylation and the various aspects of their biological significance. The substrate proteins of prenyltransferases are known to carry a sequence signal composed of a cysteine-containing 4–5 residue stretch at the utmost C-terminal end that is N-terminally preceded by a flexible and polar linker region of ca. 10 residues. Postprenylation processing of substrate proteins can involve C-terminal proteolysis, C-terminal carboxyl methylation, and other steps of maturation. The prenyl anchor functions as module for membrane attachment or for protein–protein interaction. Prenyl anchor carrying proteins fulfill a large array of functions in signaling and regulation of cellular processes. Therefore, they are involved in the pathogenesis of a variety of human diseases, the most prominent one being cancer. Farnesyltransferase inhibitors show surprisingly high efficiency in controlling tumor growth in model systems but, so far, clinical trials with human patients have remained without the desired success. Interference into prenylation pathways appears also a promising treatment principle in a variety of parasitic diseases.     

Chemical characterization of the smallest S-nitrosothiol, HSNO; cellular cross-talk of H2S and S-nitrosothiols

Dihydrogen sulfide recently emerged as a biological signaling molecule with important physiological roles and significant pharmacological potential. Chemically plausible explanations for its mechanisms of action have remained elusive, however. Here, we report that H(2)S reacts with S-nitrosothiols to form thionitrous acid (HSNO), the smallest S-nitrosothiol. These results demonstrate that, at the cellular level, HSNO can be metabolized to afford NO(+), NO, and NO(-) species, all of which have distinct physiological consequences of their own. We further show that HSNO can freely diffuse through membranes, facilitating transnitrosation of proteins such as hemoglobin. The data presented in this study explain some of the physiological effects ascribed to H(2)S, but, more broadly, introduce a new signaling molecule, HSNO, and suggest that it may play a key role in cellular redox regulation.     

Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington''s disease (HD), Alzheimer''s disease (AD), Parkinson''s disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.     

Exploring and exploiting the therapeutic potential of glycoconjugates

Carbohydrates, either bound to proteins or in lipids, play essential roles as communication molecules in many intercellular and intracellular processes. In particular, carbohydrates are important mediators of cell-cell recognition events and have been implicated in related processes such as cell signaling regulation, cellular differentiation and immune response. This diverse utility has long suggested the power of carbohydrates in therapeutic approaches. This Concepts article highlights the recent potential uses of glycoconjugates as therapeutics, with particular reference to glycopeptides, glycoproteins, glycodendrimers, and glycoarrays.     

The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family

The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.     

Infinite Assembly of Folded Proteins in Evolution,Disease, and Engineering

Mutations and changes in a protein's environment are well known for their potential to induce misfolding and aggregation, including amyloid formation. Alternatively, such perturbations can trigger new interactions that lead to the polymerization of folded proteins. In contrast to aggregation, this process does not require misfolding and, to highlight this difference, we refer to it as agglomeration. This term encompasses the amorphous assembly of folded proteins as well as the polymerization in one, two, or three dimensions. We stress the remarkable potential of symmetric homo‐oligomers to agglomerate even by single surface point mutations, and we review the double‐edged nature of this potential: how aberrant assemblies resulting from agglomeration can lead to disease, but also how agglomeration can serve in cellular adaptation and be exploited for the rational design of novel biomaterials.     

Signalling Properties of Inositol Polyphosphates

Several studies have identified specific signalling functions for inositol polyphosphates (IPs) in different cell types and have led to the accumulation of new information regarding their cellular roles as well as new insights into their cellular production. These studies have revealed that interaction of IPs with several proteins is critical for stabilization of protein complexes and for modulation of enzymatic activity. This has not only revealed their importance in regulation of several cellular processes but it has also highlighted the possibility of new pharmacological interventions in multiple diseases, including cancer. In this review, we describe some of the intracellular roles of IPs and we discuss the pharmacological opportunities that modulation of IPs levels can provide.     

Designing Molecular Spanners to Throw in the Protein Networks

Proteins govern most aspects of cellular life and, through specific interfaces, are typically involved in intricate protein–protein interaction (PPI) networks and signaling pathways. Subtle up- or downregulation of key protein functions and PPIs results in disease; still, the preferred option to contrast the role of a protein in disease and healthy conditions alike remains its outright shutdown through orthosteric ligands that block its active site. Here, we explore subtler alternatives to modulate proteins and PPIs. Driven by a view of proteins as dynamic entities, we discuss ways to identify allosteric binding sites, which, when targeted by tailored ligands, can induce significant changes in the active site of a protein, and lead to agonistic or antagonistic effects. We also summarize the selective regulation of specific PPIs—either direct or allosteric—and show that effects can be stabilizing as well as destabilizing, depending on how the conformational equilibrium of a protein is shifted.     

磷酸化蛋白质组学中的分离富集方法研究进展

蛋白质的磷酸化是一种可逆性的翻译后修饰,在细胞的增值、分化、信号转导以及转录与翻译调控、蛋白质复合体的形成、蛋白质降解等方面发挥着极为重要的作用.因此磷酸化蛋白的鉴定成为翻译后修饰研究的重要内容.但由于磷酸化蛋白的丰度较低, 难以用质谱直接检测.为了解决这个问题,改善质谱对磷酸肽的信号响应, 需要对磷酸化蛋白质或磷酸肽进行富集.本文系统地介绍了磷酸化蛋白组学研究中应用较为广泛和最新建立的各种分离富集方法的原理、特点、应用研究进展,包括抗体富集法、激酶特异富集法、亲和富集法、化学修饰法、多种色谱分离富集方法以及MALDI靶盘富集法.     

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

Direct Modulation of Small GTPase Activity and Function

Small GTPases are a family of GDP‐/GTP‐binding proteins that serve as biomolecular switches inside cells to control a variety of essential cellular processes. Aberrant function and regulation of small GTPases is associated with a variety of human diseases, thus rendering these proteins highly interesting targets in drug discovery. However, this class of proteins has been considered “undruggable”, as intensive decade‐long efforts did not yield clinically relevant direct modulators of small GTPases. Recently, the targeting of small GTPases has gained fresh impetus through the discovery of novel transient cavities on the protein surfaces and the application of new targeting strategies. Besides Ras proteins, other small GTPases have attracted increased attention since improved biological insight in combination with novel targeting strategies identified them as promising targets in drug discovery. This Review gives an overview of relevant aspects of the superfamily of small GTPases and summarizes recent progress and perspectives for the direct modulation of these challenging targets.     

Autophagy regulation by acetylation—implications for neurodegenerative diseases

Posttranslational modifications of proteins, such as acetylation, are essential for the regulation of diverse physiological processes, including metabolism, development and aging. Autophagy is an evolutionarily conserved catabolic process that involves the highly regulated sequestration of intracytoplasmic contents in double-membrane vesicles called autophagosomes, which are subsequently degraded after fusing with lysosomes. The roles and mechanisms of acetylation in autophagy control have emerged only in the last few years. In this review, we describe key molecular mechanisms by which previously identified acetyltransferases and deacetylases regulate autophagy. We highlight how p300 acetyltransferase controls mTORC1 activity to regulate autophagy under starvation and refeeding conditions in many cell types. Finally, we discuss how altered acetylation may impact various neurodegenerative diseases in which many of the causative proteins are autophagy substrates. These studies highlight some of the complexities that may need to be considered by anyone aiming to perturb acetylation under these conditions.Subject terms: Acetylation, Alzheimer''s disease     

5-Hydroxymethylcytosine, the sixth base of the genome

5-Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5-methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection--most notably LC-MS and glucosyltransferase assays--helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue-specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis. Evidence is currently accumulating that hmC may not exclusively be an intermediate of an active demethylation process, but that it functions instead as an important epigenetic marker.     

Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins

Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.