Volume 386, Issue 8

Pre-mRNA splicing is catalyzed by two unique spliceosomes, designated U2- or U12-dependent. In contrast to the well-characterized U2-dependent spliceosome, much remains to be learned about the less abundant U12-type spliceosome. This review focuses on recent advances in elucidating the structure and function of the minor U12-dependent spliceosome. Interesting similarities and differences between the U12- and U2-dependent spliceosomes are also highlighted.

The proteasome is a compartmentalized, ATP-dependent protease composed of more than 30 subunits that recognizes and degrades polyubiquitinated substrates. Despite its physiological importance, many aspects of the proteasome's structural organization and regulation remain poorly understood. In addition to the proteins that form the proteasome holocomplex, there is increasing evidence that proteasomal function is affected by a wide variety of associating proteins. A group of ubiquitin-binding proteins assist in delivery of substrates to the proteasome, whereas proteasome-associated ubiquitin ligases and deubiquitinating enzymes may alter the dynamics of ubiquitin chains already associated with the proteasome. Some proteins appear to influence the overall stability of the complex, and still others have the capacity to activate or inhibit the hydrolytic activity of the core particle. The increasing number of interacting proteins identified suggests that proteasomes, as they exist in the cell, are larger and more diverse in composition than previously assumed. Thus, the study of proteasome-associated proteins will lead to new perspectives on the dynamics of this uniquely complex proteolytic machine.

Cell survival under severe thermal stress requires the activity of a bi-chaperone system, consisting of the ring-forming AAA+ chaperone ClpB (Hsp104) and the DnaK (Hsp70) chaperone system, which acts to solubilize and reactivate aggregated proteins. Recent studies have provided novel insight into the mechanism of protein disaggregation, demonstrating that ClpB/Hsp104 extracts unfolded polypeptides from an aggregate by threading them through its central pore. This translocation activity is necessary but not sufficient for aggregate solubilization. In addition, the middle (M) domain of ClpB and the DnaK system have essential roles, possibly by providing an unfolding force, which facilitates the extraction of misfolded proteins from aggregates.

The flexibility of chromatin that enables translation of environmental cues into changes in genome utilisation, relies on a battery of enzymes able to modulate chromatin structure in a highly targeted and regulated manner. The most dynamic structural changes are brought about by two kinds of enzymes with different functional principles. Changes in the acetylation status of histones modulate the folding of the nucleosomal fibre. The histone-DNA interactions that define the nucleosome itself can be disrupted by ATP-dependent remodelling factors. This review focuses on recent developments that illustrate various strategies for integrating these disparate activities into complex regulatory schemes. Synergies may be brought about by consecutive or parallel action during the stepwise process of chromatin opening or closing. Tight co-ordination may be achieved by direct interaction of (de-)acetylation enzymes and remodelling ATPases or even permanent residence within the same multi-enzyme complex. The fact that remodelling ATPases can be acetylated by histone acetyltransferases themselves suggests exciting possibilities for the co-ordinate modulation of chromatin structure and remodelling enzymes.

Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo . In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.

Triple-helical collagen IV protomers associate through their N- and C-termini, forming a three-dimensional network that provides basement membranes with mechanical strength. Within this network, the C-terminal non-collagenous (NC1) domains form tight dimeric junctions. Crystallographic analyses of isolated NC1 domains show two trimeric cap-like structures interacting via a large interface. Previously, for NC1 from human placenta type-IV collagen we described covalent α1-α1 and α2-α2 crosslinks between Met93 and Lys211 of opposing α1(IV) and α2(IV) NC1-chains, which further stabilize this interface and explain the occurrence of reduction-insensitive NC1-chain dimers. However, their existence was recently questioned, and we therefore analyzed NC1-domain dimers in more detail by biochemical and protein crystallographic methods. Short-exposure diffraction data show a clear electron density cross-connecting the respective residues, which gradually disappears with prolonged crystal irradiation. Sequence analyses of isolated tryptic peptides derived from denatured NC1 monomers and dimers indicate that only the dimers, but not the monomers, yield these chemically labile cross-linked peptides. These data clearly demonstrate the presence of reduction-resistant, but chemically and radiation-sensitive covalent crosslinks between the side chains of Met93 and Lys211 in human placenta type-IV collagen.

For the first time we have extracted, solubilized and identified growth factors, such as insulin growth factor II (IGF-II), bone morphogenetic protein-2 (BMP-2), and transforming growth factor-β (TGF-β), from archaeological compact human bone and tooth dentin dating from the late pre-ceramic pottery Neolithic (late PPNB) and the early Middle Ages. These factors are typical of special physiological or pathological situations in the metabolism of bone. The extracellular matrix proteins from bone and teeth of individuals from the late PPNB and early Middle Ages were separated by 2-D electrophoresis and more than 300 different protein spots were detected by silver staining. The matrix protein patterns of compact bone and tooth from the same individual (early Middle Ages) are very different and only 16% of the protein spots were detected in both compact bone and tooth dentin.

We demonstrate that basic components of the plastid protein-import apparatus originally found in pea, Toc34, Toc159, and Tic110, are also conserved in evolutionarily younger gymnosperms. We show that multiple isoforms of the preprotein receptor Toc34 differentially accumulate in various stages of needle development, while the amounts of Toc159 drastically decrease during chloroplast morphogenesis. Spruce Toc34 and Toc159 receptors are able to recognise and interact with the angiosperm precursor of the Rubisco small subunit. Young proplastids found in closed buds contain a highly elevated number of protein translocation complexes equipped with only two types of outer envelope receptors, Toc159 and a 30-kDa Toc34-related protein. Photosystem II (PSII) can already be assembled in a fully functional complex at this very early stage of needle development, suggesting that no additional receptor isoforms are needed for translocation of all necessary PSII components. We conclude that the accumulation of evolutionarily conserved plastid preprotein translocation components is differentially regulated during spruce needle development.

The kidney is a target organ for thyroid hormone action and a variety of renal transport processes are altered in response to impaired thyroid functions. To investigate the effect of thyroid hormone on the expression of the renal proximal tubular high-affinity-type H + -peptide cotransporter (PEPT2) in rats, hypothyroidism was induced in animals by administration of methimazole (0.05%) via drinking water. After 7 weeks of treatment, hypothyroidism was confirmed by determining serum free T 3 and free T 4 concentrations. Northern blotting was used to examine the expression of PEPT2 mRNA in kidney tissues from hypothyroid rats compared to control rats. Hypothyroidism resulted in an increased level of total renal PEPT2 mRNA (121.1±3.3% vs. control 100±2.8%; p =0.008). The mRNA results were confirmed by immuno-blotting, which demonstrated significantly increased protein levels (162% vs. control 100%; p <0.01). Immunohistochemistry also revealed increased PEPT2 protein levels in the proximal tubules of treated compared to non-treated rats. In summary, PEPT2 is the first proximal tubule transporter protein that shows increased expression in states of hypothyreosis. As PEPT2 reabsorbs filtered di- and tripeptides and peptide-like drugs, the present findings may have important implications in nutritional amino acid homeostasis and for drug dynamics in states of altered thyroid function.

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of α-helical conformation, as measured by circular dichroism. A similar effect was observed when a His 6 -tag was linked to the C-terminus of Sapp1p or its precursor.

Neutrophil elastase (NE) and cathepsin G (CG), the proteolytic enzymes localized in azurophil granules of neutrophils (PMN), are involved in PMN responses to various stimuli. When released at sites of inflammation, they participate in the degradation of numerous proteins involved in the regulation of the immune response. In this study, we employed ADAM17 -/- fibroblasts stably transfected with cDNA of human pro-tumor necrosis factor α (proTNFα) (ADAM17 -/- TNF + ) to investigate the effects of NE and CG on shedding and degradation of TNFα. Both NE and CG were found to diminish the level of membrane TNFα (mTNFα) as measured by flow cytometry. This process was accompanied by the accumulation of biologically active soluble TNFα (sTNFα) in the culture medium, as determined by an increase in both the cytotoxic activity of TNFα and its ability to serve as a co-stimulator in the induction of inducible nitric oxide synthase (iNOS). However, in contrast to CG, NE at high concentrations was able to degrade sTNFα released from the cell surface. Using soluble recombinant human TNFα, we identified Val 93 -Ala 94 and Val 117 -Glu 118 as the NE cleavage sites within the sTNFα molecule. Taken together, the ability of NE and CG to modulate levels of membrane and soluble forms of TNFα may contribute to the proinflammatory activity of neutrophils.