Transpeptidation and reverse proteolysis and their consequences for immunity

Reverse proteolysis and transpeptidation lead to the generation of polypeptide sequences that cannot be inferred directly from genome sequences as they are post-translational phenomena. These phenomena have so far received little attention although the physiological consequences may reach far. The protease-mediated synthesis of several immunodominant MHC class I antigens was recently reported, underscoring its importance to immunity. Reverse proteolytic and transpeptidation mechanisms as well as conditions that favor successful protease-catalyzed synthetic events are discussed here.     

Calcium-dependent proteolysis of calcium-binding proteins

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.     

A Stereochemical Model of the Transpeptidation Complex

Abstract

Molecular models are proposed to describe the relative arrangement of aminoacyl and peptidyl tRNAs when bound to their respective A. and P sites on the ribosome. The crystallographically determined structures of tRNA^asp and tRNA^phe have served as the models for these bound structures, while the imposed steric constraints for the model complexes were based on the results of published experimental data. The constructed models satisfy the stereochemical requirements needed for codon-anticodon interaction and for peptide bond formation. In this paper, the results of the complex containing tRNA^phe as the A. and P site bound transfer RNAs, is compared to a similarly constructed model which uses tRNA^lsp as the ribosome-bound transfer RNAs. The models have the following three major features: 1) the aminoacyl and peptidyl transfer RNAs assume an angle of approximately 45° relative to each other; 2) in providing the proper stereochemistry for peptide bond condensation, a significant kink must be present in the messenger RNA between the A. site and P site codons; and 3) a comparison of the two model complexes indicates that structural variations between the tRNAs or any allosteric transitions of the transfer RNAs associated with codon-anticodon recognition may be accommodated in the model by way of freedom of rotation about the phosphate backbone bonds in the mRNA between consecutive codons.     

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain

Meiosis reinitiation in starfish oocytes is characterized by Ca(2+) transients in the cytosol and in the nucleus and is accompanied by the disassembly of the nuclear envelope, a process which is likely to be mediated by the cleavage of selected proteins. We have used mass spectrometry analysis (mass profile fingerprinting) on 2D polyacrylamide gels of extracts of oocytes in which meiosis resumption was induced by 1-methyladenine and have identified five proteins that were specifically degraded: alpha-tubulin, lamin B, dynamin, and two kinds of actin. They are all components of the cytoskeleton or associated with it. We then investigated whether calpain, which is activated by the increase in cell Ca(2+), could cleave the same proteins that became degraded under the influence of 1-methyladenine and thus be involved in nuclear membrane breakdown. The investigation was prompted by the finding that microinjection of calpain into the nuclei of prophase arrested oocytes induced meiosis in the absence of 1-methyladenine. Incubation of prophase arrested (disrupted) oocytes with calpain produced a 2D gel protein pattern in which some of the degradation products coincided with those seen in oocytes challenged with 1-methyladenine.     

Cell cycle-regulated proteolysis of mitotic target proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.     

Inhibition of ubiquitin-dependent proteolysis by non-ubiquitinatable proteins

The effect in reticulocyte lysates of proteins with blocked amino groups on the ATP-dependent degradation of casein and serum albumin was studied in order to assess the extent to which proteins with blocked and with free amino groups share common paths of proteolytic degradation. Completely acetylated or succinylated casein and acetylated or succinylated serum albumin (reduced and carboxymethylated), in addition to other amino-modified proteins, inhibited the ATP-dependent proteolysis of both casein and reduced carboxymethylated serum albumin. Inhibition of serum albumin degradation by acetylated serum albumin was competitive, whereas inhibition of casein degradation by acetylated casein was largely competitive with evidence of mixed kinetics. The different amino-blocked proteins studied, which were largely unfolded under assay conditions, were similarly effective as inhibitors on a weight basis, with Ki values in the range 0.2-0.6 mg/ml; there was no correlation between the ability of the blocked proteins to serve as proteolysis substrates and their effectiveness as inhibitors. Studies of the effects of acetylated proteins on the conjugation of ubiquitin to serum albumin and casein demonstrated that the acetylated proteins blocked formation of ubiquitin-albumin conjugates and of selected casein conjugates; the steady state concentration of selected conjugates of endogenous lysate proteins was increased in the presence of amino-blocked proteins. The results suggest that proteins with blocked amino groups, which cannot serve as substrates for ubiquitin conjugation, can compete for binding to those ubiquitin conjugation factors that recognize and ubiquitinate potential substrates of the ubiquitin pathway. The similar inhibitory properties of the different blocked proteins in turn suggest that a common factor in binding to these conjugation factors may be recognition of the polypeptide backbone.     

Probing the high energy states in proteins by proteolysis

Unless the native conformation has an unstructured region, proteases cannot effectively digest a protein under native conditions. Digestion must occur from a higher energy form, when at least some part of the protein is exposed to solvent and becomes accessible by proteases. Monitoring the kinetics and denaturant dependence of proteolysis under native conditions yields insight into the mechanism of proteolysis as well as these high-energy conformations. We propose here a generalized approach to exploit proteolysis as a tool to probe high-energy states in proteins. This "native state proteolysis" experiment was carried out on Escherichia coli ribonuclease HI. Mass spectrometry and N-terminal sequencing showed that thermolysin cleaves the peptide bond between Thr92 and Ala93 in an extended loop region of the protein. By comparing the proteolysis rate of the folded protein and a peptidic substrate mimicking the sequence at the cleavage site, the energy required to reach the susceptible state (Delta G(proteolysis)) was determined. From the denaturant dependence of Delta G(proteolysis), we determined that thermolysin digests this protein through a local fluctuation, i.e. localized unfolding with minimal change in solvent assessable surface area. Proteolytic susceptibilities of proteins are discussed based on the finding of this local fluctuation mechanism for proteolysis under native conditions.     

Context-dependent resistance to proteolysis of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.     

Dislocation of membrane proteins in FtsH-mediated proteolysis.

Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.     

Down-regulation of model yeast proteins by ubiquitin-dependent proteolysis

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a multitude of proteins. I will attempt to bring together our recent data on the down-regulation of two yeast model proteins, the galactose transporter Gal2 and fructose-1,6-bisphosphatase, by ubiquitin-dependent proteolysis triggered by the addition of easily fermentable carbon sources.     

Changes in teleost yolk proteins during oocyte maturation: correlation of yolk proteolysis with oocyte hydration

Yolk proteins of prematuration occytes and postmaturation eggs were compared by SDS gel electrophoresis in several teleosts, including freshwater species that produce demersal eggs, estuarine and marine species with demersal eggs, and marine species with pelagic eggs. In certain teleosts distinct changes in yolk protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins at this time; proteolysis is most pronounced in marine fishes with pelagic eggs. In many teleosts the oocyte swells by hydration during maturation; this hydration is also most pronounced in marine fishes with pelagic eggs. The extent of yolk proteolysis is well correlated with the extent of oocyte hydration during maturation.     

重组人胰岛素制备过程中转肽工艺的研究

目的：酵母表达体系制备重组人胰岛素的工艺中,转肽反应属于酶促半合成反应,过程复杂,成本高昂,通过实验研究提高反应效率,降低反应成本。方法：通过优化转肽反应中胰蛋白酶和双保护苏氨酸（Thr（But）OBut）的比例,寻找有利的反应条件,为进一步的条件确定奠定基础;从提高转肽反应物的反应效率出发,调整工艺顺序,将一步转肽反应调整为两步转肽反应;以胰蛋白酶和双保护苏氨酸两种反应物,进行综合成本分析比较;同时采用药典方法测定两种不同工艺制备的重组人胰岛素,进行生物活性的比较分析。结果：通过实验研究,一步转肽反应的收率最高可以达到74%,而两步转肽收率的收率最高可以达到90%;两步转肽反应相比一步转肽反应,酶量减少70%,双保护苏氨酸的使用量降低62.5%,同时提高了转肽反应中胰岛素原的反应浓度,达到12.5 mM,有着进一步开发的潜质;生物活性测定两种工艺生产的重组人胰岛素均符合中国药典的要求。结论：两步转肽反应对比一步转肽反应,提高了反应效率,产物的纯度较高,降低了反应的成本,有利于提高国产胰岛素的市场竞争力。