Ubiquitin-proteasome system

The capacity for exquisite regulation of ubiquitylation provides eukaryotic cells with a means to fine-tune both protein function and levels. This complex set of processes affects myriad proteins and potentially impacts all cellular processes. Ubiquitylation is brought about through multienzyme processes, with specificity conferred primarily by interactions of substrates with specific ubiquitin protein ligases (E3s) in association with ubiquitin conjugating enzymes (E2s). Regulation of ubiquitylation occurs at multiple levels, including E2-E3 interactions, substrate recognition, chain elongation, binding of ubiquitin to conserved motifs and deubiquityation. This review presents the fundamentals of the ubiquitin conjugating system.     

Expanding role of molecular chaperones in regulating α-synuclein misfolding; implications in Parkinson’s disease

Protein misfolding under stressful environmental conditions cause several cellular problems owing to the disturbed cellular protein homeostasis, which may further lead to neurological disorders like Parkinson’s disease (PD), Alzheimer’s disease (AD), Amyloid lateral sclerosis and Huntington disease (HD). The presence of cellular defense mechanisms like molecular chaperones and proteasomal degradation systems prevent protein misfolding and aggregation. Molecular chaperones plays primary role in preventing protein misfolding by mediating proper native folding, unfolding and refolding of the polypeptides along with vast number of cellular functions. In past few years, the understanding of molecular chaperone mechanisms has been expanded enormously although implementation to prevent protein aggregation diseases is still deficient. We in this review evaluated major classes of molecular chaperones and their mechanisms relevant for preventing protein aggregation, specific case of α-synuclein aggregation. We also evaluate the molecular chaperone function as a novel therapeutic approach and the chaperone inhibitors or activators as small molecular drug targets.     

CAS proteins in normal and pathological cell growth control

Proteins of the CAS (Crk-associated substrate) family (BCAR1/p130Cas, NEDD9/HEF1/Cas-L, EFS/SIN and CASS4/HEPL) are integral players in normal and pathological cell biology. CAS proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation, and have more recently been linked to a role in progenitor cell function. Reflecting these complex functions, over-expression of CAS proteins has now been strongly linked to poor prognosis and increased metastasis in cancer, as well as resistance to first-line chemotherapeutics in multiple tumor types including breast and lung cancers, glioblastoma, and melanoma. Further, CAS proteins have also been linked to additional pathological conditions including inflammatory disorders, Alzheimer’s and Parkinson’s disease, as well as developmental defects. This review will explore the roles of the CAS proteins in normal and pathological states in the context of the many mechanistic insights into CAS protein function that have emerged in the past decade.     

Sumoylation regulates diverse biological processes

Ten years after its discovery, the small ubiquitin-like protein modifier (SUMO) has emerged as a key regulator of proteins. While early studies indicated that sumoylation takes place mainly in the nucleus, an increasing number of non-nuclear substrates have recently been identified, suggesting a wider stage for sumoylation in the cell. Unlike ubiquitylation, which primarily targets a substrate for degradation, sumoylation regulates a substrate’s functions mainly by altering the intracellular localization, protein-protein interactions or other types of post-translational modifications. These changes in turn affect gene expression, genomic and chromosomal stability and integrity, and signal transduction. Sumoylation is counter-balanced by desumoylation, and well-balanced sumoylation is essential for normal cellular behaviors. Loss of the balance has been associated with a number of diseases. This paper reviews recent progress in the study of SUMO pathways, substrates, and cellular functions and highlights important findings that have accelerated advances in this study field and link sumoylation to human diseases. Received 19 March 2007; received after version 16 July 2007; accepted 1 August 2007     

Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3–substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein–protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.     

The role of deubiquitinating enzymes in apoptosis

It has become apparent that ubiquitination plays a critical role in cell survival and cell death. In addition, deubiquitinating enzymes (DUBs) have been determined to be highly important regulators of these processes. Cells can be subjected to various stresses and respond in a variety of different ways ranging from activation of survival pathways to the promotion of cell death, which eventually eliminates damaged cells. The regulatory mechanisms of apoptosis depend on the balanced action between ubiquitination and deubiquitination systems. There is a growing recognition that DUBs play essential roles in regulating several binding partners to modulate the process of apoptosis. Thus, the interplay between the timing of DUB activity and the specificity of ubiquitin attachment and removal from its substrates during apoptosis is important to ensure cellular homeostasis. This review discusses the role of a few ubiquitin-specific DUBs that are involved in either promoting or suppressing the process of apoptosis.     

Glycogen synthase kinase 3: A key regulator of cellular fate

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified as a key regulator of insulin-dependent glycogen synthesis. GSK-3 was subsequently shown to function in a wide range of cellular processes including differentiation, growth, motility and apoptosis. Aberrant regulation of GSK-3 has been implicated in a range of human pathologies including Alzheimer’s disease, non-insulin-dependent diabetes mellitus (NIDDM) and cancer. As a consequence, the regulation of GSK-3 and the therapeutic potential of GSK-3 inhibitors have become key areas of investigation. This review will focus on the mechanisms of GSK-3 regulation, with emphasis on modulation by upstream signals, control of substrate specificity and GSK-3 localisation. The details of these mechanisms will be discussed in the context of specific signalling pathways. Received 30 January 2007; received after revision 5 March 2007; accepted 16 April 2007     

Cellular factors modulating the mechanism of tau protein aggregation

Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones.     

Sorting receptor SORLA: cellular mechanisms and implications for disease

Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor that directs cargo proteins, such as kinases, phosphatases, and signaling receptors, to their correct location within the cell. The activity of SORLA assures proper function of cells and tissues, and receptor dysfunction is the underlying cause of common human malignancies, including Alzheimer’s disease, atherosclerosis, and obesity. Here, we discuss the molecular mechanisms that govern sorting of SORLA and its cargo in multiple cell types, and why genetic defects in this receptor results in devastating diseases.     

Ubiquitin-proteasome system

The yeast Saccharomyces cerevisiae has turned out to be an invaluable tool in the molecular biological sciences for elucidating the housekeeping functions of eukaryotic cells. Due to its easy amenability to biochemical, genetic, molecular biological and cell biological experimentation, including genomics and proteomics, yeast has become one of the most frequently used eukaryotic model organisms. One of the fields where studies in yeast have a truly pacemaking character is cellular control by proteolysis. The function of vacuolar (lysosomal) proteolysis was elucidated. The in vivo role of ubiquitin and its relation to the proteasome was uncovered. This research led to an avalanche of studies in many different eukaryotic systems, including mammals, and provided us with surprising new insights in cellular control in health and disease.     

Plexin structures are coming: opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions

Plexin transmembrane receptors and their semaphorin ligands, as well as their co-receptors (Neuropilin, Integrin, VEGFR2, ErbB2, and Met kinase) are emerging as key regulatory proteins in a wide variety of developmental, regenerative, but also pathological processes. The diverse arenas of plexin function are surveyed, including roles in the nervous, cardiovascular, bone and skeletal, and immune systems. Such different settings require considerable specificity among the plexin and semaphorin family members which in turn are accompanied by a variety of cell signaling networks. Underlying the latter are the mechanistic details of the interactions and catalytic events at the molecular level. Very recently, dramatic progress has been made in solving the structures of plexins and of their complexes with associated proteins. This molecular level information is now suggesting detailed mechanisms for the function of both the extracellular as well as the intracellular plexin regions. Specifically, several groups have solved structures for extracellular domains for plexin-A2, -B1, and -C1, many in complex with semaphorin ligands. On the intracellular side, the role of small Rho GTPases has been of particular interest. These directly associate with plexin and stimulate a GTPase activating (GAP) function in the plexin catalytic domain to downregulate Ras GTPases. Structures for the Rho GTPase binding domains have been presented for several plexins, some with Rnd1 bound. The entire intracellular domain structure of plexin-A1, -A3, and -B1 have also been solved alone and in complex with Rac1. However, key aspects of the interplay between GTPases and plexins remain far from clear. The structural information is helping the plexin field to focus on key questions at the protein structural, cellular, as well as organism level that collaboratoria of investigations are likely to answer.     

Molecular pathology and pathobiology of osteoarthritic cartilage

The biochemical properties of articular cartilage rely on the biochemical composition and integrity of its extracellular matrix. This matrix consists mainly of a collagen network and the proteoglycan-rich ground substance. In osteoarthritis, ongoing cartilage matrix destruction takes place, leading to a progressive loss in joint function. Beside the degradation of molecular matrix components, destabilization of supramolecular structures such as the collagen network and changes in the expression profile of matrix molecules also take place. These processes, as well as the pattern of cellular reaction, explain the pathology of osteoarthritic cartilage degeneration. The loss of histochemical proteoglycan staining reflects the damage at the molecular level, whereas the supramolecular matrix destruction leads to fissuring and finally to the loss of the cartilage. Chondrocytes react by increasing matrix synthesis, proliferating, and changing their cellular phenotype. Gene expression mapping in situ and gene expression profiling allows characterization of the osteoarthritic cellular phenotype, a key determinant for understanding and manipulating the osteoarthritic disease process.     

Mechanisms of cellular invasion by intracellular parasites

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world’s population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite–host cell interactions, forming the basis of the parasite’s cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.     

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate. Received 7 November 2005; received after revision 7 January 2006; accepted 13 January 2006     

Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, β- and γ-secretases is a determining factor in Alzheimer’s disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aβ production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aβ production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.     

Molecular targets of non-steroidal anti-inflammatory drugs in neurodegenerative diseases

During the last decade, interest has grown in the beneficial effects of non-steroidal anti-inflammatory drugs (NSAIDs) in neurodegeneration, particularly in pathologies such as Alzheimer’s (AD) and Parkinson’s (PD) disease. Evidence from epidemiological studies has indicated a decreased risk for AD and PD in patients with a history of chronic NSAID use. However, clinical trials with NSAIDs in AD patients have yielded conflicting results, suggesting that these drugs may be beneficial only when used as preventive therapy or in early stages of the disease. NSAIDs may also have salutary effects in other neurodegenerative diseases with an inflammatory component, such as multiple sclerosis and amyotrophic lateral sclerosis. In this review we analyze the molecular (cyclooxygenases, secretases, NF-κB, PPAR, or Rho-GTPasas) and cellular (neurons, microglia, astrocytes or endothelial cells) targets of NSAIDs that may mediate the therapeutic function of these drugs in neurodegeneration. Received 4 December 2006; received after revision 24 January 2007; accepted 23 February 2007