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Procitani radovi (Radovi koje mi zadao Alessandro ("Proteasome…

- - - - Proteins associated with nuclear functions, such as cyclins, cyclin-
        dependent kinase inhibitors, and transcription factors (NF-κB, IκB and p53), were among the first physiological proteasomal substrates to be identified [4,5]. Subsequently, cytoplasmic proteins whose turnover was also dependent on the proteasome were identified. Among these cytosolic proteins are newly synthesized proteins that do not reach their intended conformation or location
    - - More than thirty different subunits are present in the mature
        RP-CP assemblies. The CP contains seven distinct α and seven distinct β subunits. These subunits are arranged into seven-membered rings
        that are stacked to give a barrel-shaped particle with α7β7β7α7 con- figuration. The active site residues reside in the CP cavity formed by both β rings.
        
        The α rings are responsible for gating the central channels on both
        sides of the CP barrel. Normally, the α ring gates are in a closed confor mation. Thus, the freeCP has latent enzyme activity.Opening of the α ring requires the RP,which consists of two subcomplexes, the base and the lid.
        
        The RP base contains six different ATPase subunits (Rpt1 to Rpt6; RP triphosphatase) andfour different non-ATPase subunits (Rpn1, Rpn2, Rpn10 and Rpn13: RP non-triphosphatase).
        
        The six-membered ATPase ring of the RP base, which is adjacent to the CP α ring, is responsible for the ATP hydrolysis that promotes unfolding of the sub- strate and its translocation into the CP cavity.
        
        Rpn10, a more distant RP base subunit, recognizes the poly-ubiquitin chain that marks a pro- tein for proteasomal degradation.
        
        The lid contains at least nine non-ATPase subunits.
        
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    - - It is now clear that proteasomes are largely nuclear in di-
        viding cells and have the ability to move between the cyto- and nucleoplasm to carry out protein degradation at the right place and at the right time
        
        Early studies localized proteasomes primarily to the nuclei of Xenopus laevis oocytes and cultured mammalian cells
        
        Other studies, however, showed that proteasomes change their intracellular localization in cultured mammalian cells depending on the cell cycle stage [23]. Proteasomes were observed to accumulate at the nuclear periphery during mitosis and to be predominantly nuclear in
        daughter cells after cytokinesis. During interphase, proteasomes were
        seen in clusters in the cytoplasm and at the nuclear matrix
        
        The first in vivo localization studies of GFP-labeled proteasomes in
        budding and fission yeasts showed that most proteasomes, either CP (la-
        beled by α4 and β5) or RP (labeled by Rpt1, Rpt4 and Rpn11), were nu-
        clear and decorated the nuclear membranes, which at the cytoplasmic
        side merge with the endoplasmic reticulum [29–31]. At the same time,
        these observations were surprising as it had been anticipated that
        proteasomes would be predominantly localized in the cytoplasm. Mean-
        while imaging studies firmly established that proteasomes are primarily
        nuclear in logarithmically growing yeast cells, independently of which
        proteasomal subunit was labeled with GFP
        
        Live cell imaging studies showed that proteasomes are also primarily nuclear, in rapidly proliferating mammalian cells.
        
        Previous studies showed that the nuclear periphery is the major site of
        proteasome-mediated proteolysis
        
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      - Nuclear import of proteasomes in dividing cells
        
        Our subsequent observations from reconstitution assays supported these previous findings that mature
        proteasomes are hardly transported through nuclear pores by the classi-
        cal NLS receptor pathway . In the classical NLS receptor pathway,
        which is highly conserved from yeast to human, the NLS cargo is recog-
        nized by the heterodimeric receptor, importin/karyopherin αβ.
        
        Karyopherin α binds the NLS cargo, while karyopherin β mediates an in-
        teraction with the phenylalanine-glycine rich nuclear pore proteins that
        are present along the passage through the nuclear pore. On the nucleo-
        plasmic side of the nuclear pore, the transport complex encounters the
        small GTPase, Ran. Ran in its GTP-bound form displaces the NLS cargo
        and karyopherin α by binding to karyopherin β [46]. Intriguingly, not
        only is the mature CP nuclear, but its inactive precursor complexes are
        also primarily nuclear, suggesting that the nuclear CP is matured at its destination
        
        Inactive precursor complexes of the CP are referred to as half-CP.
        These complexes contain one α ring, the CP-dedicated chaperone,
        Ump1, and β subunits with propeptides. Ump1 guides the dimerization
        of two half-CPs into the pre-holo-CP . Within the pre-holo-CP, the β
        propeptides are cleaved off by autocatalysis and Ump1 is degraded by
        the nascent CP
        
        In 1990, Keiji Tanaka proposed a model for nuclear import of the CP.
        He hypothesized that two CP conformations exist, one import-
        competent in which the NLSs are exposed, and one import-
        incompetent in which the NLSs are masked [38]. In precursor com-
        plexes, the NLSs are apparently exposed and facilitate nuclear import
        of the precursor complexes by the classical NLS-dependent pathway
        . The β subunits, which lack NLSs, are piggybacked with the α ring
        into the nucleus. In the mature CP, the α ring is closed and the NLSs
        seem to be less accessible, which hinders nuclear import of the mature
        CP.
        
        In budding yeast, the CP precursor complexes, the RP lid, and the
        RP base seem to be imported separately into the nucleus by the
        NLS-dependent pathway [39,56]. Rpt2 and Rpn2 confer NLSs to the
        RP base. Since the RP base is assembled mainly from two precur
        or complexes, one containing Rpt2 and the other one containing
        Rpn2, it is possible that assembly of the RP base occurs in the nucleus
        
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    - - Most of the cells of our body, especially neuronal cells, do not divide
        and instead arrest in the G0 stage of the cell cycle. Yeast cells that are
        grown to stationary phase enter a G0-like stage referred to as quies-
        cence. During the transition of yeast cells from logarithmic growth to
        stationary phase, proteasomes migrate from the nuclear matrix to the
        nuclear envelope. Although the significance of proteasome movement
        is not yet clear, it could reflect changes in proteolytic demands. In quies-
        cent cells, proteasomes accumulate in one or two dot-like clusters on
        the cytoplasmic side of the nuclear envelope (Fig. 1, right panels). This
        phenotype was studied in detail by Sagot and co-workers who
        coined the term proteasome storage granuli (PSG) for these dot-like
        clusters. PSG are not surrounded by membranes and move freely through the cytoplasm.
        
        They appear to pinch off the nuclear envelope,
        when proteasomes are exported from the nucleus into the cytoplasm
        upon the transition from proliferation to quiescence. During prolonged
        quiescence, proteasomes are retained in the PSG and the nucleus re-
        mains deficient in proteasomes . The question arises as to what is
        the “molecular glue” that holds proteasomes together within the PSG.
        Upon exit of cells from quiescence, this “molecular glue” resolves, PSG
        rapidly clear and proteasomes relocate to the nucleus within a few mi-
        nutes. Thus, PSG were assumed to serve as storage depot during quies-
        cence.
        
        Motile dot-like clusters are also observed at the nuclear periphery
        upon chemical inhibition of the proteasome in mammalian cells or in
        temperature-sensitive proteasomal yeast mutants grown at restrictive
        temperature. Like proteasome inhibitors, these proteasomal mutations
        affect the protein degradation of cell cycle regulators and cause cell
        cycle arrest. Although a genetically or chemically induced cell cycle ar-
        rest is not equivalent to quiescence, inhibited proteasomes appear to
        be sequestered into so-called juxta nuclear quality control compart-
        ments (JUNQs) that are located at the cytoplasmic side of the nuclear
        envelope. Since poly-ubiquitylated misfolded proteins were detected
        in JUNQ, JUNQ was proposed to represent a major site of proteasomal
        proteolysis.
        
        Intriguing-
        ly, GFP-labeled mammalian proteasomes are clustered in cytosolic
        dot-like structures in neuronal cells [69]. Local needs of coordinated
        protein degradation for synaptic plasticity seem to recruit proteasomes
        from the dendritic shaft into dot-like structures of the spines. .
        Recently N-myristoylation of Rpt2, an RP base ATPase, was reported to
        regulate proteasome localization. Proteasomes without N-myristoylation
        seem to leak out off the nucleus and to be sequestered into dot-like struc-
        tures in the cytoplasm
    - - This suggests that the mechanisms for nuclear import in
        Xenopus eggs and quiescent yeast cells differ. Nonetheless, both cell systems have in common that a rapid nuclear import of preassembled CPs
        is required upon the resumption of cell division.
        
        Finally I want to project the studies in quiescent yeast cells to
        G0-arrested mammalian cells in which proteasomes also accumulate
        in reversible and motile clusters [69]. I anticipate that proteasome
        clusters will protect against neurodegenerative disorders, as long
        as these clusters remain mobile and reversible. Co-localizations of
        proteasomes with ubiquitin-positive protein inclusions are often
        considered as hallmarks of many diseases like Parkinson's,
        Alzheimer's and Huntington's [8]. In such cases, the burden of
        ubiquitylated proteins probably exceeds the degradation capacity of the proteasome. This is similar to the situation provoked in cul-
        tured cells that are exposed to proteasome inhibitors or that over-
        express proteasomal substrates. The intriguing question is what factors transform soluble proteasome clusters into cytotoxic protein
        aggregates, so-called aggresomes [77]. Post-translational modifications related with aging may be the reason for the irreversibility and immobility of protein clusters. These irreversible and immobile protein clusters may finally lead to disease and death of quiescent
        cells, unless autophagic processes are activated to remove them.
  - - - Proteasome particles are formed by the dynamic association of
        several sub-complexes, a 20S core particle (20S CP), either single or associated with one or two regulatory particles (RPs) of identical or different protein composition. Proteasome complexes thus display a high degree of heterogeneity.
        
        The 20S CP presents a a7b7b7a7 barrel-like structure and was shown to exist in the eukaryotic cell as four different subtypes, depending on the subsets of incorporated catalytic beta subunits. In the standard proteasome (sP20S), the two b rings each contain three standard catalytic subunits, b1, b2, and b5, which are replaced by distinct immunosubunits, b1i, b2i, and b5i, in the immunoproteasome (iP20S), respectively. Two intermedi- ate 20S CP subtypes, b5i 20S proteasome (b5i P20S) and b1ib5i 20S proteasome (b1ib5i P20S), bearing a mixed incorporation of standard and immunosubunits, b1, b2, b5i and b1i, b2, b5i, respectively, have also been identified in a wide range of cell types and tissues (Guillaume et al, 2010). / a=alpha, b=beta , for proteasome subunit composition)
        
        The catalytic subunits are responsible for the three proteasome proteolytic activities (trypsin like, chymotrypsin like, and caspase like), which can be modulated by the replacement of standard subunits by immunosubunits.
        
        The iP20S is induced during the immune response in mammals but also exists in various amounts as constitutive proteasome complexes, depending on tissues or cell type
        
        One 20S CP can interact at its two sides with either two identical regulators or two different ones, thus forming hybrid proteasomes (Tanahashi et al, 2000). The most studied regulator, the 19S RP, is involved in the recognition, the unfolding, and the translocation of poly-ubiquitinated substrates into the 20S CP for degradation. In addition to the 19S RP, PA28ab and PA28c RPs are abundant 20S proteasome-associated regulators pres- ent in the cytosol and the nucleus, respectively (Drews et al, 2007; Fabre et al, 2013). They catalyze protein degradation through an ubiquitin-independent pathway, which still needs to be completely clarified (Stadtmueller
    - - System-wide studies of the composition and dynamics of protein complexes have recently been addressed using an alternative method to AP-MS, protein correlation profiling associated with mass spectrometry (PCP-MS).
        
        This approach involves biochemical fractionation proce- dures and allows the assignment of proteins to specific organelles (Andersen et al, 2003; Foster et al, 2006; Gatto et al, 2010) or, more recently, to protein complexes (Kristensen et al, 2012), by comparing the proteins elution profiles acquired by quantitative MS.
        
        As far as interactome studies are concerned, PCP-MS increases the analysis throughput of protein complexes dynamics (Kristensen et al, 2012) and also helps monitoring the impact of subunit isoforms or post-translational modifications in multiprotein complexes (Kirkwood et al, 2013). Interestingly, the heterogeneity of protea- somes can, at least in part, be resolved using PCP-MS on size exclu- sion chromatography (SEC)-separated protein complexes because this approach is able to distinguish and quantify the relative propor- tions of singly and doubly capped 20S CPs
        
        By correlating proteins abundances across a large set of 24 proteasome samples immunopurified from nine different human cell lines, we observed that the two main 20S proteasome subtypes, sP20S and iP20S, interact with a different subset of regulators. Some of these preferential interactions were validated by artificially or physiologi- cally changing the proportions of both 20S CP subtypes in assem- bled proteasomes.
        
        This result therefore suggests that sP20S and iP20S interact with a different subset of proteins, in particular with distinct regulators. In proteasome immunoprecipitates, the b5 subunit is indeed highly correlated with PI31 and PA200 but not with the PA28ab and PA28c complexes. On the other hand, the b2i subunit correlated well with the 20S protea- some-associated PA28ab complex but very badly with PA200 and PI31. Conversely, the 19S regulator does not correlate with any of the two 20S proteasome subtypes as it exhibits almost identical coefficients of determination for both the sP20S. Its abundance is rather correlated with the one of the ncP20S , which represents all 20S proteasome forms. This means that the 19S RP associates as well with the sP20S as with the iP20S.
        
        In total, 60 putative PIPs correlate (R > 0.8) with the sP20S and 31 do with the iP20S in the proteasome immunoprecipitates (Fig 4C). These proteins therefore constitute possible substrates or PIPs associating preferentially with one particular 20S proteasome subtype.
        
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  - - - The folded structures of proteins are only marginally stable, and subtle changes due to mutation may tip the balance (2). Furthermore, a substan- tial fraction of proteins (15 to 30%of mammalian proteomes) lack ordered structure partially or entirely (3), and the formation of toxic aggregates by such metastable proteins is associated with Alzheimer’sand Parkinson’s disease. Thus, protein quality control and the maintenance of proteome balance are critical for cellular and organismal health.
        
        To ensure proteostasis, organisms from all domains of life invest in an extensive quality control network, integrating molecular chaperones, which mediate protein folding and conformational repair, with the ubiquitin-proteasome system (UPS) and autophagy, which remove terminally misfolded proteins and aggregates. Importantly, the capacity of the proteostasis network (PN) declines during aging (4), facilitating the emergence of chronic diseases caused by protein aggregation, including neurodegeneration, type II diabetes, heart disease, and certain forms of cancer.
        
        Whereas small proteins up to ~100 aa may
        fold rapidly (within milliseconds) and with full yield in vitro (8), folding is often inefficient for larger proteins, owing to off-pathway aggrega- tion. Proteins >100 aa constitute the major frac- tion of all proteomes, with large multidomain proteins strongly increasing in number from prokaryotes to eukaryotes
        
        The folding of such proteins in vivo is further compounded by macromolecular crowding (300 to 400 g of total protein per liter in the cytosol) (9), which enhances the tendency of folding intermediates and misfolded states to aggregate. A major func- tion of the chaperone network is to prevent such
        aberrant interactions, which are often irreversible. We define amolecular chaperone as any protein that interacts with, stabilizes, or helps another protein to acquire its functionally active conforma- tion,without being present in its final structure