Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting

Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.     

MHC molecules protect T cell epitopes against proteolytic destruction.

There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures that some of the Ag is salvaged? Using a cell-free system we demonstrate that an Ag fragment, once bound to a MHC class II molecule, is effectively protected against proteolytic destruction by cathepsin B and pronase E. The bound fragment, however, can be modified by aminopeptidase N. We suggest that MHC class II molecules play an important regulatory role in the physiologic processing of Ag.     

Pituitary endopeptidases

This review summarizes our knowledge of pituitary endopeptidases. Emphasis has been placed on well-characterized enzymes and their potential roles in proteolytic processes of the pituitary. Because of space limitations, degradation of biologically active peptide by crude preparations has generally not been discussed. Only a few proteolytic enzymes are at present adequately characterized, and knowledge of their physiological function in vivo is insufficient. Among the many functions of proteolytic enzymes, those that are specific for the pituitary as an endocrine gland are of primary interest. Such functions include inactivation of neuropeptides and factors that control the secretory function of the pituitary, processing of precursors destined for secretion, selective cleavage of prohormones into active fragments, and degradation of inactive fragments. While some of the enzymes described here, such as cathepsin D, could be expected to have primarily a degradative function, others could potentially be involved in hormonal metabolism, since they exhibit trypsin-like, chymotrypsin-like, and dipeptidyl carboxypeptidase-like activities, all potentially useful in hormonal conversions. Data suggestive of the presence in the pituitary of enzymes involved in removal of the 'signal sequence', and enzymes involved in hormone processing by cleavage of bonds after a pair of basic residues and in the subsequent removal of these residues by a carboxypeptidase B-like activity have been published. None of these enzymes, however, has been isolated or purified to a degree that would allow determination of its specificity, mechanisms of action, physicochemical properties, and susceptibility to specific inhibitors. Questions that remain unresolved ask whether differences in the processing pathways in various anatomical parts of the pituitary are due to the presence of proteases with different specificities, or to different disposition of these enzymes, and factors, such as conformation of the substrate and its secondary modification, for example by glycosylation or phosphorylation. Proof of a functional involvement of a protease in hormonal processing should include demonstration that inhibition of activity results in inhibition of processing in the intact cell. Specific inhibitors of processing enzymes could potentially be used to modulate pituitary function, and thus have pharmacological interest. Although there are few answers to the above problems at present, the questions are well defined, and it can be expected that the rapidly expanding research on pituitary proteases will soon provide some of the answers.     

Participation of proteolytic enzymes in the interaction of plants with phytopathogenic microorganisms

Different forms of participation of proteolytic enzymes in pathogenesis and plant defense are reviewed. Together with extracellular proteinases, phytopathogenic microorganisms produce specific effectors with proteolytic activity and are able to act on proteins inside the plant cell. In turn, plants use both extracellular and intracellular proteinases for defense against phytopathogenic microorganisms. Among the latter, a special role belongs to vacuolar processing enzymes (legumains), which perform the function of caspases in the plant cell.     

Nonhuman cells correctly sort and process the human lysosomal enzyme cathepsin D

Cathepsin D, like most lysosomal enzymes, undergoes multiple proteolytic cleavages during its lifetime. Although the significance of the earliest cleavages of cathepsin D is apparent (loss of the NH2-terminal signal peptide and activation peptide), functions of the two later cleavages are not understood and do not occur in all species. To examine these later events, a cDNA coding for human cathepsin D, which is normally processed to a two-chain form, was isolated and then expressed in mammalian cells from species which do not process the enzyme to the two-chain form. Analysis of the expressed human cathepsin D demonstrated proteolytic processing identical with that seen in normal human fibroblasts. Since processing to the two-chain form of the enzyme occurs in the lysosome, these studies revealed that the human cathepsin D was correctly sorted. The data also indicated that the sorting mechanism was conserved between diverse species and that late proteolytic processing in a variety of species was not determined by the presence or absence of the processing enzymes in the cell.     

Protein degradation in MHC class II antigen presentation: opportunities for immunomodulation

Proteolysis is required for two steps of the MHC class II antigen-processing pathway, degradation of invariant chain and cleavage of protein antigens. Invariant chain dissociation from MHC is limited by a final proteolytic event which is tightly regulated in both temporal and tissue-specific ways. In contrast, enzymes involved in antigen proteolysis remain ill-defined. Gene 'knockout' experiments of housekeeping proteolytic enzymes suggest either that these enzymes do not play a major role, or that antigen proteolysis is too degenerate for this type of analysis. The possible role of two other proteinases, cathepsin E and aspariginyl endopeptidase is discussed. Finally, the data implicating antigen processing in repertoire generation is briefly considered. We conclude that selective regulation of endosomal proteolysis could have profound implications for control of immunity against infection, as well as in autoimmunity.     

Structures at the proteolytic processing region of cathepsin D

The amino acid sequences at the "proteolytic processing regions" of cathepsin Ds have been determined for the enzymes from cows, pigs, and rats in order to deduce the sites of cleavage as well as the function of the proteolytic processing of cathepsin D. For bovine cathepsin D, the "processing region" sequence was determined from a peptide isolated from the single-chain enzyme. The COOH-terminal sequence of the light chain and the NH2-terminal sequence of the heavy chain were also determined. The processing region sequence of porcine cathepsin D was determined from its cDNA structure, and the same structure from rat cathepsin D was determined from the peptide sequence of the single-chain rat enzyme. From sequence homology to other aspartic proteases whose x-ray crystallographic structures are known, such as pepsinogen and penicillopepsin, it is clear that the processing regions are insertions to form an extended beta-hairpin loop between residues 91 and 92 (porcine pepsin numbers). However, the sizes of the processing regions of cathepsin Ds from different species are considerably different. For the enzymes from rats, cows, pigs, and human, the sizes of the processing regions are 6, 9, 9, and 11 amino acid residues, respectively. The amino acid sequences within the processing regions are considerably different. In addition, the proteolytic processing sites were found to be completely different in the bovine and porcine cathepsin Ds. While in the porcine enzyme, an Asn-Ser bond and a Gly-Val bond are cleaved to release 5 residues as a consequence of the processing; in the bovine enzyme, two Ser-Ser bonds are cleaved to release 2 serine residues. These findings would argue that the in vivo proteolytic processing of the cathepsin D single chain is probably not carried out by a specific "processing protease." Model building of the cathepsin D processing region conformation was conducted utilizing the homology between procathepsin D and porcine pepsinogen. The beta-hairpin structure of the processing region was found to (i) interact with the activation peptide of the procathepsin D in a beta-structure and (ii) place the Cys residue in the processing region within disulfide linkage distance to Cys-27 of cathepsin D light chain. These observations support the view that the processing region of cathepsin D may function to stabilize the conformation of procathepsin D and may play a role in its activation.     

Secretion of somatostatin by Saccharomyces cerevisiae. Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes

Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease. Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide. Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin. To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed. Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor. No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site. Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted. Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor. We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors. This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.     

A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing.     

Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe- AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.     

Protein degradation within mitochondria: versatile activities of AAA proteases and other peptidases

Cell survival depends on essential processes in mitochondria. Various proteases within these organelles regulate mitochondrial biogenesis and ensure the complete degradation of excess or damaged proteins. Many of these proteases are highly conserved and ubiquitous in eukaryotic cells. They can be assigned to three functional classes: processing peptidases, which cleave off mitochondrial targeting sequences of nuclearly encoded proteins and process mitochondrial proteins with regulatory functions; ATP-dependent proteases, which either act as processing peptidases with regulatory functions or as quality-control enzymes degrading non-native polypeptides to peptides; and oligopeptidases, which degrade these peptides and mitochondrial targeting sequences to amino acids. Disturbances of protein degradation within mitochondria cause severe phenotypes in various organisms and can lead to the induction of apoptotic programmes and cell-specific neurodegeneration in mammals. After an overview of the proteolytic system of mitochondria, we will focus on versatile functions of ATP-dependent AAA proteases in the inner membrane. These conserved proteolytic machines conduct protein quality surveillance of mitochondrial inner membrane proteins, mediate vectorial protein dislocation from membranes, and, acting as processing enzymes, control ribosome assembly, mitochondrial protein synthesis, and mitochondrial fusion. Implications of these functions for cell-specific axonal degeneration in hereditary spastic paraplegia will be discussed.     

Novel aspects of the apolipoprotein-E receptor family: regulation and functional role of their proteolytic processing

Studies related to the functional and regulatory aspects of proteolytic processing are of interest to cell biologists,developmental biologists and investigators who work on human diseases.Much of what ...     

Correct proteolytic cleavage is required for the cell adhesive function of uvomorulin

All Ca2(+)-dependent cell adhesion molecules are synthesized as precursor polypeptides followed by a series of posttranslational modifications including proteolytic cleavage. The mature proteins are formed intracellularly and transported to the cell surface. For uvomorulin the precursor segment is composed of 129-amino acid residues which are cleaved off to generate the 120-kD mature protein. To elucidate the role of proteolytic processing, we constructed cDNAs encoding mutant uvomorulin that could no longer be processed by endogenous proteolytic enzymes and expressed the mutant polypeptides in L cells. Instead of the recognition sites for endogenous proteases, these mutants contained either a recognition site of serum coagulation factor Xa or a new trypsin cleavage site. The intracellular proteolytic processing of mutant polypeptides was inhibited in both cases. The unprocessed polypeptides were efficiently expressed on the cell surface and had other features in common with mature uvomorulin, such as complex formation with catenins and Ca2(+)-dependent resistance to proteolytic degradation. However, cells expressing unprocessed polypeptides showed no uvomorulin-mediated adhesive function. Treatment of the mutant proteins with the respective proteases results in cleavage of the precursor region and the activation of uvomorulin function. However, other proteases although removing the precursor segment were ineffective in activating the adhesive function. These results indicate that correct processing is required for uvomorulin function and emphasize the importance of the amino-terminal region of mature uvomorulin polypeptide in the molecular mechanism of adhesion.     

(Pro)Insulin processing

Insulin, the major secreted product of the β-cells of the islets of Langerhans, is initially synthesized as a precursor (preproinsulin), from which the mature hormone is excised by a series of proteolytic cleavages. This review provides a personal narrative of some of the key research projects leading to the identification of the central processing enzymes as proprotein convertase 1, proprotein convertase 2, and carboxypeptidase E. It also discusses the central roles of the intragranular environment and chaperone-like proteins in modulating processing activity.     

Effective extraction of astaxanthin pigment from shrimp using proteolytic enzymes

The present study was to investigate efficient extraction conditions of astaxanthin from shrimp wastes for utilizing it as a functional food additive. In order to enhance the stability of pigments, proteolytic enzymes were applied to extract astaxanthin as carotenoprotein. Also, various conditions such as acid ensilaging of samples, using EDTA solution and adding various enzymes were examined to optimize extraction processing. After extraction, all of the extracts were partitioned in a separate funnel and each astaxanthin content was analyzed by using UV/VIS spectrophotometer. Carotenoprotein was effectively extracted from non-acid ensilaged shrimp wastes by using EDTA medium and a proteolytic enzyme. In that case, the reddish top layer showed 91.9% of recovery and the blackish bottom layer did 2.3% and its separation ratio was about 0.2 (v/v); therefore concentration and purification of reddish top layer were more desirable than those of whole extract.     

Proteolysis of β-endorphin in brain tissue

The processing of β-endorphin by brain enzymes into peptides related to the behaviorally active γ- and α-type endorphins and the sequence of proteolytic events in the conversion process are described. Multiple enzyme activities contribute to the generation of the peptides with neurotropic activity. It is proposed that the processing into γ- and α-type neuropeptides is a post-secretional event. The enzymes involved may have a key role in the nature and levels of neurotropic β-endorphin fragments in the brain.     

Mitochondrial processing peptidases

Three peptidases are responsible for the proteolytic processing of both nuclearly and mitochondrially encoded precursor polypeptides targeted to the various subcompartments of the mitochondria. Mitochondrial processing peptidase (MPP) cleaves the vast majority of mitochondrial proteins, while inner membrane peptidase (IMP) and mitochondrial intermediate peptidase (MIP) process specific subsets of precursor polypeptides. All three enzymes are structurally and functionally conserved across species, and their human homologues begin to be recognized as potential players in mitochondrial disease.     

Identification of proteases with shared functions to the proprotein processing protease Krp1 in the fission yeast Schizosaccharomyces pombe

Many secretory proteins are synthesized as inactive proproteins that undergo proteolytic activation as they travel through the eukaryotic secretory pathway. The best characterized family of processing enzymes are the prohormone convertases or kexins, and these are responsible for the processing of a wide variety of prohormones and other precursors. Recent work has identified other proteases that appear to be involved in proprotein processing, but characterization of these enzymes is at an early stage. Krp1 is the only kexin identified in the fission yeast Schizosaccharomyces pombe, in which it is essential for cell viability. We have used a genetic screen to identify four proteases with specificities that overlap Krp1. Two are serine proteases, one is a zinc metalloprotease (glycoprotease) and one is an aspartyl protease that belongs to the recently described yapsin family of processing enzymes. All four proteases support the growth of a yeast strain lacking Krp1, and each is able to process the P-factor precursor, the only substrate currently known to be processed by Krp1.     

大鵟消化系统蛋白水解酶种类和活性分析

采用蛋白水解酶复性电泳(G-PAGE)技术对大(Buteo hemilasius)消化系统5种器官腺胃、胰脏、十二指肠、空肠、大肠蛋白水解酶的种类和性质进行了研究,以期为研究野生鸟类的分类地位、系统演化提供基础资料,结果表明,①受pH值的影响和制约,大消化系统蛋白水解酶的活性在碱性、中性与酸性条件下递减;②在酸性条件下,45 ku蛋白水解酶存在于除腺胃外的各受检器官;③pH 7.0时,腺胃、胰脏酶谱相似,均含有683、5、342、0 ku的蛋白水解酶;④pH 8.0时,空肠和十二指肠的蛋白水解酶种类最多、活性最强,分别检出8种和7种蛋白水解酶。总之,pH值对蛋白水解酶的活性有明显的制约作用,46、41ku蛋白水解酶随着pH值的增高而失去活性,为酸性蛋白水解酶,250、2064、5 ku蛋白水解酶随着pH值的增高活性逐渐增强,为碱性蛋白水解酶。十二指肠和空肠的蛋白水解酶种类多、活性强,可能为蛋白质消化的主要场所。     

Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells

The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We have used affinity-purified antisera that recognize only the products of POMC processing and immunoelectron microscopy to identify the compartments of rat pituitary corticotropes and mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling of cryostat sections and immunogold labeling of ultrathin frozen sections were used for localization of the processing sites. By both procedures we detected processed peptides in Golgi cisternae and secretion granules. Within the Golgi, labeling was limited to the last or transmost cisterna and was most concentrated in its dilated rims which contain condensing secretory protein. No labeling of other Golgi cisternae was seen. All Golgi cisternae were labeled, however, when antisera that recognize unprocessed POMC were used for immunolabeling. We conclude that in AtT-20 and rat pituitary cells: 1) processing of POMC through at least two endo- and exoproteolytic cleavage steps and alpha-amidation of joining peptide begin in the trans Golgi subcompartment; 2) no detectable processing takes place before POMC reaches the trans Golgi cisterna; and 3) this Golgi cisterna as well as secretion granules contain the active enzymes necessary for proteolytic processing and alpha-amidation.