Cellular maintenance of nuclear protein homeostasis

The accumulation and aggregation of misfolded proteins is the primary hallmark for more than 45 human degenerative diseases. These devastating disorders include Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. Over 15 degenerative diseases are associated with the aggregation of misfolded proteins specifically in the nucleus of cells. However, how the cell safeguards the nucleus from misfolded proteins is not entirely clear. In this review, we discuss what is currently known about the cellular mechanisms that maintain protein homeostasis in the nucleus and protect the nucleus from misfolded protein accumulation and aggregation. In particular, we focus on the chaperones found to localize to the nucleus during stress, the ubiquitin–proteasome components enriched in the nucleus, the signaling systems that might be present in the nucleus to coordinate folding and degradation, and the sites of misfolded protein deposition associated with the nucleus.     

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ER–Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER–Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER–Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER–Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ER–Golgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS.     

UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97

The highly conserved AAA ATPase Cdc48/p97 acts on ubiquitylated substrate proteins in cellular processes as diverse as the fusion of homotypic membranes and the degradation of misfolded proteins. The 'Ubiquitin regulatory X' (UBX) domain-containing proteins constitute the so far largest family of Cdc48/p97 cofactors. UBX proteins are involved in substrate recruitment to Cdc48/p97 and in the temporal and spatial regulation of its activity. In combination with UBX-like proteins and other cofactors, they can assemble into a large variety of Cdc48/p97-cofactor complexes possessing distinct cellular functions. This review gives an overview of the different subfamilies of UBX proteins and their functions, and discusses general principles of Cdc48/p97 regulation by these cofactors.     

Beiträge zum Stickstoffmetabolismus grüner Pflanzen II. Der Einfluss exogener Glucose auf die Aufnahme von Nitrat und Ammonium aus der Nährlösung und auf den Proteingehalt vonSpirodela oligorrhiza

Summary The uptake of ammonium and nitrate by normal and glucose-stimulatedSpirodela oligorrhiza plants was investigated. Nitrate is used more rapidly by plants grown on glucose than by controls, suggesting a stimulation of nitrate assimilation by glucose. In plants growing on a normal nutrient, the contents of both water-soluble and water-insoluble proteins linearly increase with time and nitrogen uptake. In glucose-stimulated cultures soluble protein increases rapidly during the first 10 days of cultivation, but declines thereafter, while insoluble protein rises continuously over the whole period of time. The decreasing ratio of soluble and insoluble protein reflects an alteration of the protein and enzyme pattern, which is suggested to be the primary reason for the change of plastid ultrastructure and the loss of photosynthetic activity.

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Die Arbeiten wurden vom Schweizerischen Nationalfonds unterstützt.     

Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins

By virtue of their general ability to bind (hold) translocating or unfolding polypeptides otherwise doomed to aggregate, molecular chaperones are commonly dubbed “holdases”. Yet, chaperones also carry physiological functions that do not necessitate prevention of aggregation, such as altering the native states of proteins, as in the disassembly of SNARE complexes and clathrin coats. To carry such physiological functions, major members of the Hsp70, Hsp110, Hsp100, and Hsp60/CCT chaperone families act as catalytic unfolding enzymes or unfoldases that drive iterative cycles of protein binding, unfolding/pulling, and release. One unfoldase chaperone may thus successively convert many misfolded or alternatively folded polypeptide substrates into transiently unfolded intermediates, which, once released, can spontaneously refold into low-affinity native products. Whereas during stress, a large excess of non-catalytic chaperones in holding mode may optimally prevent protein aggregation, after the stress, catalytic disaggregases and unfoldases may act as nanomachines that use the energy of ATP hydrolysis to repair proteins with compromised conformations. Thus, holding and catalytic unfolding chaperones can act as primary cellular defenses against the formation of early misfolded and aggregated proteotoxic conformers in order to avert or retard the onset of degenerative protein conformational diseases.     

Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy

Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin–proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα–γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407–4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα–γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.     

Many mechanisms, one entrance: membrane protein translocation into the nucleus

The inner nuclear membrane harbors a unique set of membrane proteins, many of which interact with nuclear intermediate filaments and chromatin components and thus play an important role in nuclear organization and gene expression regulation. These membrane proteins have to be constantly transported into the nucleus from their sites of synthesis in the ER to match the growth of the nuclear membrane during interphase. Many mechanisms have evolved to enable translocation of these proteins to the nucleus. The full range of mechanisms goes from rare autophagy events to regulated translocation using the nuclear pore complexes. Though mechanisms involving nuclear pores are predominant, within this group an enormous mechanistic range is observed from free diffusion through the peripheral channels to many distinct mechanisms involving different nucleoporins and other components of the soluble protein transport machinery in the central channels. This review aims to provide a comprehensive insight into this mechanistic diversity.     

Hsp70 in mitochondrial biogenesis: From chaperoning nascent polypeptide chains to facilitation of protein degradation

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix.     

Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response

Protein quality control is vital for all living cells and sophisticated molecular mechanisms have evolved to prevent the excessive accumulation of unfolded proteins. High-temperature requirement A (HtrA) proteases have been identified as important ATP-independent quality-control factors in most species. HtrA proteins harbor a serine-protease domain and at least one peptide-binding PDZ domain to ensure efficient removal of misfolded or damaged proteins. One distinctive property of HtrAs is their ability to assemble into complex oligomers. Whereas all examined HtrAs are capable of forming pyramidal 3-mers, higher-order complexes consisting of up to 24 molecules have been reported. Tight control of chaperone and protease function is of pivotal importance in preventing deleterious HtrA-protease activity. In recent years, structural biology provided detailed insights into the molecular basis of the regulatory mechanisms, which include unique intramolecular allosteric signaling cascades and the dynamic switching of oligomeric states of HtrA proteins. Based on these results, functional models for many family members have been developed. The HtrA protein family represents a remarkable example of how structural and functional diversity is attained from the assembly of simple molecular building blocks.     

The evolution of domain arrangements in proteins and interaction networks

Proteins are composed of domains, which are conserved evolutionary units that often also correspond to functional units and can frequently be detected with reasonable reliability using computational methods. Most proteins consist of two or more domains, giving rise to a variety of combinations of domains. Another level of complexity arises because proteins themselves can form complexes with small molecules, nucleic acids and other proteins. The networks of both domain combinations and protein interactions can be conceptualised as graphs, and these graphs can be analysed conveniently by computational methods. In this review we summarise facts and hypotheses about the evolution of domains in multi-domain proteins and protein complexes, and the tools and data resources available to study them.Received 20 September 2004; received after revision 23 October 2004; accepted 1 November 2004     

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc₃Man₇GlcNAc₂ FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.     

Periostin and its interacting proteins in the construction of extracellular architectures

Periostin is a matricellular protein that is composed of a multi-domain structure with an amino-terminal EMI domain, a tandem repeat of four FAS 1 domains, and a carboxyl-terminal domain. These distinct domains have been demonstrated to bind to many proteins including extracellular matrix proteins (Collagen type I and V, fibronectin, tenascin, and laminin), matricellular proteins (CCN3 and βig-h3), and enzymes that catalyze covalent crosslinking between extracellular matrix proteins (lysyl oxidase and BMP-1). Adjacent binding sites on periostin have been suggested to put the interacting proteins in close proximity, promoting intermolecular interactions between each protein, and leading to their assembly into extracellular architectures. These extracellular architectures determine the mechanochemical properties of connective tissues, in which periostin plays an important role in physiological homeostasis and disease progression. In this review, we introduce the proteins that interact with periostin, and discuss how the multi-domain structure of periostin functions as a scaffold for the assembly of interacting proteins, and how it underlies construction of highly sophisticated extracellular architectures.     

Aggrecan, aging and assembly in articular cartilage

The primary function of articular cartilage to act as a self-renewing, low frictional material that can distribute load efficiently at joints is critically dependent upon the composition and organisation of the extracellular matrix. Aggrecan is a major component of the extracellular matrix, forming high molecular weight aggregates necessary for the hydration of cartilage and to meet its weight-bearing mechanical demands. Aggregate assembly is a highly ordered process requiring the formation of a ternary complex between aggrecan, link protein and hyaluronan. There is extensive age-associated heterogeneity in the structure and molecular stoichiometry of these components in adult human articular cartilage, resulting in diverse populations of complexes with a range of stabilities that have implications for cartilage mechanobiology and integrity. Recent findings have demonstrated that aggrecan can form ligands with other matrix proteins. These findings provide new insights into mechanisms for aggregate assembly and functional protein networks in different cartilage compartments with maturation and aging.     

Cellular uptake of amelogenin,and its localization to CD63, and Lamp1-positive vesicles

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes’ processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation. Received 27 September 2006; received after revision 24 November 2006; accepted 5 December 2006     

nDsbD: a redox interaction hub in the Escherichia coli periplasm

DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a ‘redox hub’ with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners. Received 3 February 2006; received after revision 1 March 2006; accepted 5 April 2006     

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 2002 RID="*" ID="*"Corresponding author.     

A central role for calcineurin in protein misfolding neurodegenerative diseases

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca²⁺) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca²⁺ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca²⁺ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca²⁺/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.     

Syntrophin proteins as Santa Claus: role(s) in cell signal transduction

Syntrophins are a family of cytoplasmic membrane-associated adaptor proteins, characterized by the presence of a unique domain organization comprised of a C-terminal syntrophin unique (SU) domain and an N-terminal pleckstrin homology (PH) domain that is split by insertion of a PDZ domain. Syntrophins have been recognized as an important component of many signaling events, and they seem to function more like the cell’s own personal ‘Santa Claus’ that serves to ‘gift’ various signaling complexes with precise proteins that they ‘wish for’, and at the same time care enough for the spatial, temporal control of these signaling events, maintaining overall smooth functioning and general happiness of the cell. Syntrophins not only associate various ion channels and signaling proteins to the dystrophin-associated protein complex (DAPC), via a direct interaction with dystrophin protein but also serve as a link between the extracellular matrix and the intracellular downstream targets and cell cytoskeleton by interacting with F-actin. They play an important role in regulating the postsynaptic signal transduction, sarcolemmal localization of nNOS, EphA4 signaling at the neuromuscular junction, and G-protein mediated signaling. In our previous work, we reported a differential expression pattern of alpha-1-syntrophin (SNTA1) protein in esophageal and breast carcinomas. Implicated in several other pathologies, like cardiac dys-functioning, muscular dystrophies, diabetes, etc., these proteins provide a lot of scope for further studies. The present review focuses on the role of syntrophins in membrane targeting and regulation of cellular proteins, while highlighting their relevance in possible development and/or progression of pathologies including cancer which we have recently demonstrated.