Proteolytic degradation of tyrosine nitrated proteins

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.     

Visualizing interaction of proteins relevant to Alzheimer's disease in intact cells

To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimer's disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid beta (Abeta) through sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Abeta production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein-protein-interaction in intact cells, which has proven to be a valuable method in AD research.     

Proteolytic degradation of heterologous proteins expressed inAspergillus niger

Summary Proteolytic degradation of heterologous proteins expressed in the filamentous fungusAspergillus niger reduces the yield of authentic target protein. The activities ofA. niger proteases are differentiated by their effects on two proteins expressed and secreted fromA. niger: hen egg-white lysozyme and porcine pancreatic phospholipase A₂.     

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases

Alzheimer's disease is characterized by neurodegeneration and deposition of betaA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer's disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.     

Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Proteolytic degradation of nuclear matrix proteins in rat liver and Zajdela ascites hepatoma

Long-term incubation of rat liver nuclei (particularly in 2M NaCl) led to a decrease in the yield of the nuclear matrix. However, the electrophoretic pattern of nuclear matrix proteins remained essentially unchanged. In Zajdela's ascites hepatoma, long-term incubation of the nuclei resulted in lamina proteolysis. The proteolysis was partly inhibited by phenylmethylsulfonyl fluoride in high concentrations.     

Proteolytic mechanisms in amyloid-beta metabolism: therapeutic implications for Alzheimer's disease

The accumulation of the amyloid-beta peptide, the main constituent of the "amyloid plaque", is widely considered to be the key pathological event in Alzheimer's disease. Amyloid-beta is produced from the amyloid precursor protein through the action of the proteases beta-secretase and gamma-secretase. Alternative cleavage of amyloid precursor protein by the enzyme alpha-secretase precludes amyloid-beta production. In addition, several proteases are involved in the degradation of amyloid-beta. This review focuses on the proteolytic mechanisms of amyloid-beta metabolism. An increasingly detailed understanding of proteolysis in both amyloid-beta deposition and clearance has identified some of these proteases as potential therapeutic targets for Alzheimer's disease. A more complex knowledge of these proteases takes us one step closer to developing "disease-modifying" therapies, but these advances also emphasize that significant challenges must be overcome before clinically effective drugs to treat Alzheimer's disease become a reality.     

Post-translational processing of beta-secretase in Alzheimer's disease

Beta-amyloid is released into the brains of Alzheimer's patients, where it aggregates and causes damage to neurons. It is cleaved proteolytically from a large transmembrane glycoprotein amyloid precursor protein by a membrane-bound protease, known as beta-secretase identified previously as the acid protease, Asp-2. We have shown previously that beta-secretase is up-regulated by increased intracellular cholesterol, and down-regulated by cholesterol biosynthesis inhibition. Here we show using mass spectrometry that discrete changes in the glycosylation and palmitoylation of beta-secretase occur when cells expressing it are treated with statins.     

Proteolytic processing and physiological regulation

Proteolytic processing is a common and effective mechanism of physiological regulation. The basic principle is a conformational change induced in the protein precursor by the post-translational proteolytic cleavage of a specific peptide bond. The extension of earlier studies of model zymogens to more complex systems of physiological regulation, using methods of both protein chemistry and molecular biology, has enormously extended knowledge of the repertoire of proteolytic processing reactions and has contributed significantly to current studies of the structure, domain organization and evolution of proteins.     

Current research approaches in downstream processing of pharmaceutically relevant proteins

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Proteolytic processing and regulation.

Many proteins, particularly proteolytic enzymes, protein hormones and neuropeptides are synthesized as inactive precursors that undergo posttranslational processing by proteolytic enzymes. The roots of current knowledge go back to the early observations of the activation of zymogens. A major advance followed the discovery of the polypreprotein, pre-pro-opiomelanolcortin and of proinsulin, and the characterization of the mammalian processing prohormone enzyme as members of the multidomain yeast kexin family. More recent applications of methods of molecular biology have greatly advanced our understanding of the nature and mode of action of these proteases.     

Proteolytic processing of presecretory proteins is required for development of biological activities in pancreatic exocrine proteins

The biological activities of pancreatic presecretory and secretory proteins synthesized in vitro were compared in studies of (a) the binding of nascent amylase to its substrate, glycogen, (b) the binding of nascent trypsinogen 1, trypsinogen 2+3, and chymotrypsinogen 1 to Sepharose-bound soybean trypsin inhibitor, and (c) the activation of nascent trypsinogen by porcine enterokinase. Nascent secretory proteins synthesized in vitro using a mRNA-dependent gel-filtered reticulocyte lysate translation system supplemented with canine pancreas rough microsomes or canine pancreas mRNA and micrococcal nuclease-treated microsomal membranes showed biological activities similar to authentic secretory proteins if oxidized glutathione was added during their synthesis. Proteins synthesized in the presence of membranes and the absence of glutathione showed significantly less biological activity due to incorrect development of conformation. Presecretory proteins synthesized in vitro with canine pancreas mRNA in the absence of microsomal membranes had little or no activity after translation in either the absence or presence of glutathione. These and previous findings (Scheele, G. A., and Jacoby, R. (1982) J. Biol. Chem. 257, 12277-12282) indicate that proteolytic removal of the NH2-terminal transport peptide is necessary to allow correct conformational development, including the formation of native disulfide bonds, which not only stabilizes the molecule but allows expression of authentic biological and probiological activity.     

Proteolytic processing of turnip yellow mosaic virus replication proteins and functional impact on infectivity

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA.