Proteomic analysis of ACTN4-interacting proteins reveals it’s a putative involvement in mRNA metabolism

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.     

The link between 20S proteasome activity and post-replication DNA repair in Saccharomyces cerevisiae

We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S proteasome maturase causes sensitivity to UV radiation. In the current report, we have extended this finding to show that mutations specifically compromising chymotrypsin-like or trypsin-like activity of 20S proteasome peptidases also result in increased UV sensitivity. We have also established that mutations affecting proteasome activity, namely ump1Delta, pre2-K108R and pup1-T20A, result in spontaneous and UV-induced mutator phenotypes. To elucidate the origin of these DNA repair phenotypes of the proteasomal mutants, we performed epistasis analysis, with respect to UV sensitivity, using yeast strains with the UMP1 deletion in different DNA repair backgrounds. We show that UMP1 is not epistatic to RAD23 and RAD2, which are involved in the nucleotide excision repair (NER) pathway. Instead, our results indicate that UMP1 as well as PUP1 and PRE2 (encoding catalytic subunits of 20S proteasome) belong to an epistatic group of genes functioning in post-replication DNA repair (PRR) and are hypostatic to RAD18, which, in complex with RAD6, plays a central role in PRR. We also show that UMP1 is epistatic to REV3 and RAD30, although the relationship of UMP1 with these genes is different.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.     

The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.     

Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins

TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull-down assays, co-immunoprecipitations and by yeast two-hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double-strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair.     

An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3 proteins

CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly.     

Purification and refolding of human alpha5-subunit (PSMA5) of the 20S proteasome, expressed as inclusion bodies in Escherichia coli

The 20S proteasome is the central enzyme of nonlysosomal protein degradation in both the cytosol and nucleus. It is composed of 28 protein subunits which are arranged into four staggered heptameric rings. The outer rings consist of alpha-subunits which are responsible for binding of proteasome activators, inhibitors, and regulators. To better characterize human alpha5-subunit (PSMA5) of the 20S proteasome, we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the human PSMA5, which was subcloned into the vector pET-22b (+), has been expressed as inclusion bodies in E. coli BL21 (DE3). To produce the native PSMA5, straightforward protocols have been developed for refolding the human PSMA5 in the presence of surfactants using dilution refolding and size-exclusion chromatography matrix refolding methods. After refolding, recovery yields of about 20% were obtained, respectively, with purity above 95%. The human PSMA5 was detected by dynamic light scattering in refolding process, and the molecular weight of the final refolded product was measured using gel filtration chromatography, which indicates that the human PSMA5 exists mainly as tetramer.     

Biochemical analysis of the 20 S proteasome of Trypanosoma brucei

We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.     

High-resolution AFM-imaging and mechanistic analysis of the 20 S proteasome.

As macromolecular protease complex, the 20 S proteasome is responsible for the degradation of cellular proteins and the generation of peptide epitopes for antigen presentation. Here, structural and functional aspects of the 20 S proteasome from Thermoplasma acidophilum have been investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR). Due to engineered histidine tags introduced at defined positions, the proteasome complex was pre-oriented at ultra-flat chelator lipid membranes allowing for high-resolution imaging by AFM. Within these two-dimensional protein arrays, the overall structure of the proteasome and the organization of individual subunits was resolved under native conditions without fixation or crosslinking. In addition, the substrate-proteasome interaction was monitored in real-time by SPR using a novel approach. Instead of following enzyme activity by product formation, the association and dissociation kinetics of the substrate-proteasome complex were analyzed during proteolysis of the polypeptide chain. By blocking the active sites with a specific inhibitor, the substrate binding step could be dissected from the degradation step thus resolving mechanistic details of substrate recognition and cleavage by the 20 S proteasome.     

Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast

The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells. The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings. The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role. Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed. Because of this result, the path of substrates into the catalytic chamber has remained enigmatic. We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast. We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel. The preferred conformation depends on the ligands present. Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation. These results shed new light on the possible path of the substrate into the proteolytic chamber.     

Selective activation of the 20 S proteasome (multicatalytic proteinase complex) by histone h3.

Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment.     

CHIP: a link between the chaperone and proteasome systems

CHIP, carboxy terminus of Hsc70 interacting protein, is a cytoplasmic protein whose amino acid sequence is highly conserved across species. It is most highly expressed in cardiac and skeletal muscle and brain. The primary amino acid sequence is characterized by 3 domains, a tetratricopeptide repeat (TPR) domain at its amino terminus, a U-box domain at its carboxy terminus, and an intervening charged domain. CHIP interacts with the molecular chaperones Hsc70-Hsp70 and Hsp90 through its TPR domain, whereas its U-box domain contains its E3 ubiquitin ligase activity. Its interaction with these molecular chaperones results in client substrate ubiquitylation and degradation by the proteasome. Thus, CHIP acts to tilt the folding-refolding machinery toward the degradative pathway, and it serves as a link between the two. Because protein degradation is required for healthy cellular function, CHIP's ability to degrade proteins that are the signature of disease, eg, ErbB2 in breast and ovarian cancers, could prove to be a point of therapeutic intervention.     

N(alpha)-acetylation and proteolytic activity of the yeast 20 S proteasome

N(alpha)-acetylation, catalyzed co-translationally with N(alpha)-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3. The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants, nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the alpha1, alpha2, alpha3, alpha4, alpha7, and beta3 subunits were acetylated with NAT1, the alpha5 and alpha6 subunits were acetylated with MAK3, and the beta4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha5 and alpha6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1 mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N(alpha)-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1 mutant than in the normal strain.     

Proteomic analysis of S-acylated proteins in human B cells reveals palmitoylation of the immune regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation.     

Processing of autophagic protein LC3 by the 20S proteasome

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; and addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.     

Mass spectrometry reveals the missing links in the assembly pathway of the bacterial 20 S proteasome

The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.     

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.