Specific Cleavage Sites Research Articles

CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells.

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 μm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 μm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.

EndoProteoFASP as a Tool to Unveil the Peptidome-Protease Profile: Application to Salivary Diagnostics.

In the quest to fully comprehend the proteolytic events leading to the generation of the salivary peptidome, we have developed a method for the sequential elution of salivary peptides throughout progressive endogenous proteolysis. By screening the time-dependent changes in the salivary peptidome we can predict the activity pattern of salivary proteases responsible for such peptide fingerprint and identify susceptible protein targets. Herein, we describe a step-by-step tutorial based on a filter-aided sample preparation (FASP) method, taking advantage of the endogenous salivary proteases armamentarium (endoProteoFASP), to produce new peptides from the salivary proteins, adding to those present in the sample at the time of collection. In this protocol, the different sets of peptides retrieved after sample elution are identified following a liquid chromatography-tandem mass spectrometry approach. The likelihood of a large set of endogenous proteases (collected from several public sources) to be responsible for the generation of such peptides can be predicted by the analysis of the cleavage site specificity by Proteasix ( http://proteasix.cs.man.ac.uk /) algorithm. The attained peptidome-protease profile can be useful to elucidate the peptidome dynamics and the proteolytic events underpinning pathophysiological phenomena taking place locally within the oral cavity. This may help clinicians to diagnose oral pathologies and develop preventive therapeutic plans.

Serum MicroRNA-150 Predicts Prognosis for Early-Stage Non-Small Cell Lung Cancer and Promotes Tumor Cell Proliferation by Targeting Tumor Suppressor Gene SRCIN1.

This integrative multistage study was aimed to identify circulating microRNAs (miRNAs) as prognostic biomarkers and investigate the treatment target for early-stage non-small cell lung cancer (NSCLC) patients. In stage I-II NSCLC patients, we screened and validated the miRNA ratio signatures predictive of prognosis in serum. In tumor, we found that the expression of miR-150 in identified miRNA signatures was also associated with survival. Increased miR-150 expression promoted NSCLC cell proliferation and migration and vice versa. Specific mRNA cleavage sites targeted by endogenous miR-150 in 3' untranslated region (UTR) of SRCIN1 was identified by utilizing our recently developed novel Stem-Loop-Array reverse-transcription polymerase chain reaction (SLA-RT-PCR) assay. The blocking action of miR-150 resulted in repressed NSCLC cell growth in vitro and knockdown of miR-150 caused substantial tumor volume reduction in vivo. Our findings suggest that miR-150 binding on specific recognition sites in 3' UTR of tumor suppressor gene SRCIN1 present a potential therapeutic target for NSCLC.

Proteomic identification of synaptic caspase substrates.

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapse-specific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses. Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states.

Conformations of the HIV-1 protease: A crystal structure data set analysis

The HIV protease is an important drug target for HIV/AIDS therapy, and its structure and function have been extensively investigated. This enzyme performs an essential role in viral maturation by processing specific cleavage sites in the Gag and Gag-Pol precursor polyproteins so as to release their mature forms. This 99 amino acid aspartic protease works as a homodimer, with the active site localized in a central cavity capped by two flexible flap regions. The dimer presents closed or open conformations, which are involved in the substrate binding and release. Here the results of the analysis of a HIV-1 protease data set containing 552 dimer structures are reported. Different dimensionality reduction methods have been used in order to get information from this multidimensional database. Most of the structures in the data set belong to two conformational clusters. An interesting observation that comes from the analysis of these data is that some protease sequences are localized preferentially in specific areas of the conformational landscape of this protein.

Purification and identification of a novel peptide derived from by-products fermentation of spiny head croaker ( Collichthys lucidus ) with antifungal effects on phytopathogens

In our previous study, spiny head croaker (Collichthys lucidus) by-products fermentation (SBF) products demonstrated plant growth promoting effects. In this study, the antimicrobial activities of SBF against plant pathogenic fungi and food-borne bacteria were evaluated. An active antifungal fraction SBF-3-1 was isolated from SBF using size exclusion chromatography and high performance liquid chromatography. Seven short peptides, i.e., LDEGW, DSFDFK, LDGW, FPDL, YNLDFK, FDGF and LDFE, plus one 22 residues peptide TFNTPAMYVAIQAVLSLYASGR were identified in SBF-3-1 by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI-Q-TOF-MS/MS). The peptide TFNTPAMYVAIQAVLSLYASGR (named as SBF-3-1p), a potential antimicrobial peptide predicted by the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php), was further characterized. The prediction showed that the generation of SBF-3-1p could be associated with the action of trypsin and glutamyl endopeptidase, two proteases with specific cleavage sites on the carboxyl side of arginine (R) and lysine (K), and on the carboxyl side of Glu (E), respectively. According to the structural modeling, SBF-3-1p contained one α-helix and two loop regions. The synthesized SBF-3-1p exhibited strong activity against plant pathogenic fungus Pestalotiopsis sp. Therefore, SBF should be a good source of antifungal peptide, and could be used as natural fungicides in agriculture.

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-Helicobacter pylori activity.

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6×His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34kDa, and its expression level was approximately 30mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.

Enhancing Interleukin-6 and Interleukin-11 receptor cleavage

Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1′ were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R.

Multiplexed Protease Specificity Profiling Using Isobaric Labeling.

The determination of a protease's cleavage site specificity is one of the major goals in degradomics. In the last years, the use of proteome-derived peptide libraries and liquid chromatography-mass spectrometry (LC-MS) in a proteomic identification of protease cleavage sites (PICS) experiment became popular for that purpose.In this chapter, we offer a step-by-step protocol for the execution of a quantitative proteomic identification of protease cleavage sites (Q-PICS) experiment, which enables the relative quantification of proteolytic events by isobaric labeling, e.g., with tandem mass tags (TMT). In this way, the cleavage site specificity and activity of a protease can be compared under different reaction conditions (e.g., buffer, pH, temperature, inhibitor). Multiplexing can further be used to analyze replicate experiments in parallel, decreasing instrument times and work effort significantly, or to perform internal controls.

Characterization of the Low-Molecular-Weight Human Plasma Peptidome.

The human plasma proteome represents an important secreted sub-proteome. Proteomic analysis of blood plasma with mass spectrometry is a challenging task. The high complexity and wide dynamic range of proteins as well as the presence of several proteins at very high concentrations complicate the profiling of the human plasma proteome. The peptidome (or low-molecular-weight fraction, LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based markers of disease. Peptides are generated by active synthesis and proteolytic processing, often yielding proteolytic fragments that mediate a variety of physiological and pathological functions. As such, degradomic studies, investigating cleavage products via peptidomics and top-down proteomicsin particular, have warranted significant research interest. However, due to their molecular weight, abundance, and solubility, issues with identifying specific cleavage sites and coverage of peptide fragments remain challenging. Peptidomics is currently focused toward comprehensively studying peptides cleaved from precursor proteins by endogenous proteases. This protocol outlines a standardized rapid and reproducible procedure for peptidomic profiling of human plasma using centrifugal ultrafiltration and mass spectrometry. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration (cellulose triacetate membrane) for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration, and temperature to facilitate recovery >95% and enrichment of the human plasma peptidome. This method serves as a comprehensive and facile process to enrich and identify a key, underrepresented sub-proteome of human blood plasma.

Alzheimer's disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio.

Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer’s disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aβ42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aβ42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aβ42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aβ42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins.

Proteolytic activity assayed by subcellular localization switching of a substrate

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80.

Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58- kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

Site-specific cleavage of acetoacetyl-CoA synthetase by legumain.

Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme and is responsible for the synthesis of cholesterol and fatty acids. We have previously shown that AACS is cleaved by legumain, a lysosomal asparaginyl endopeptidase. In this study, we attempted to determine the cleavage site of AACS. Mutagenesis analysis of AACS revealed that Asn547 is the specific cleavage site of AACS in mouse livers. The cleaved form of AACS (1-547) lost the ability to convert acetoacetate to acetoacetyl-CoA. Moreover, hydrodynamics-based gene transduction showed that overexpression of AACS (1-547) increases the protein expression of caveolin-1, the principal component of the caveolae. These results suggest that cleavage of AACS by legumain is critical for the regulation of enzymatic activity and results in gain-of-function changes.

The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors

Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme.

Biotechnology Based Process for Production of a Disulfide-Bridged Peptide.

A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.

Soluble VE‐cadherin contributes to loss of VE‐cadherin‐mediated adhesion and endothelial barrier function

Endothelial barrier breakdown in systemic inflammation and sepsis is a hallmark preceding organ failure and death in patients. Vascular endothelial (VE)‐cadherin‐mediated adhesion represents a crucial determinant of endothelial barrier integrity. Previously we demonstrated that under inflammatory conditions the disintegrin and metalloproteinase 10 (ADAM10) caused shedding of VE‐cadherin which resulted in the formation of soluble VE‐cadherin (sVE‐cadherin) in close temporal correlation of inflammation‐induced endothelial barrier disruption. Here we further characterized ADAM10‐induced VE‐cadherin shedding and tested whether recombinant sVE‐cadherin inferes VE‐cadherin mediated adhesion.First we identified possible cleavages sites of VE‐cadherin by mass spectrometry (MSM). Therefore, digestion of recombinant VE‐cadherin (rVE‐cadherin) with recombinant ADAM10 was performed. This led to the formation of sVE‐cadherin at 90 and 110kDa, as shown by coomassie stained gel an western blot, which correlated with several cleavage sites of the extracellular domain of VE‐cadherin. MSM showed only peptides up to 452 amino acids (aa) and one peptide at 503–530 aa, which also corresponds to the extracellular domains 1–5 of human VE‐cadherin. Additionally two other cleavage sites at 80–127aa and 160–175aa were observed. In the next step we performed atomic force microscopy measurements in a cell free sytem to test whether sVe‐cadherin blocked VE‐cadherin‐mediated adhesion. In these experiments both, sVE‐cadherin and rVE‐cadherin blocked VE‐cadherin‐mediated adhesion. Accordingly, application of rVE‐cadherin on Human dermal microvascular endothelial cells (HDMECs) resulted in a dose‐dependent loss of endothelial barrier functions as revealed by measurements of transendothelial electrical resistance (TER). In immunostaining of endothelial monolayers incubation with rVE‐cadherin led to intercellular gap formation.In summary our data demonstrate specific cleavage sites of VE‐cadherin by ADAM10. The resulting sVE‐cadherin fragments interfere VE‐cadherin mediated adhesion and thereby contribute to loss of endothelial barrier disruption. This may play a novel role in inflammation‐induced endothelial barrier breakdown.Support or Funding InformationThe present study was supported by grants from the German Research Foundation (DFG) to N.S. (Grant numbers: SCHL1962/2‐1 and SCHL 1962/4‐1).

Cleavage of proglucagon by the prohormone converting enzymes PCSK1 and PCSK2 is not dictated by the amino acid sequence immediately adjacent to the cleavage site

The peptide hormones glucagon, glucagon‐like peptide‐1, and glucagon‐like peptide‐2 are all derived from proglucagon (PG), a 160 amino acid polypeptide containing multiple potential cleavage sites designated by either single or paired basic amino acids. Initial cleavage of PG takes place on the C‐terminal side of the K70‐R71 dibasic site, which liberates the intermediates glicentin and major proglucagon fragment (MPGF). My laboratory is interested in examining the specificity of the prohormone cleaving enzymes PCSK1 and PCSK2 using both PG and proinsulin (PI) as model substrates. PG is a useful model because it has one cleavage site (the glicentin/MPGF junction) recognized almost equally well by both enzymes and multiple potential cleavage sites that are not recognized by either enzyme. PI is a good model because it has one cleavage site (the B chain/C peptide junction) preferentially recognized by PCSK1 and a second cleavage site (the C peptide/A chain junction) preferentially recognized by PCSK2. My lab has previously described the expression and purification of these two model substrates. However, the purification of PG has presented a number of problems because of the presence of contaminants that obscure detection and participate in co‐precipitation during early stages of purification. I have now devised an improved, two‐step scheme using step elution from SP Sepharose as a capture step, followed by gradient elution from Q Sepharose as the major purification step. This method resolves the problems posed by the contaminants and generates PG samples suitable for analysis of the PCSK‐mediated cleavage reaction. An in vitro cleavage assay was then set up so that the PG substrate (~3 μM) incubated with either PCSK1 or PCSK2 (~100 nM at pH 6.0 or 5.4, respectively) results in ~30% cleavage at the glicentin/MPGF junction using either enzyme. As noted above, in contrast to the situation with PG, the normal maturation pathway for PI exhibits a marked preference for PCSK1‐mediated cleavage at the B chain/C peptide junction. To examine whether this cleavage selectivity is due to amino acids in the immediate vicinity of the cleavage site, the sequence EAED from the C‐terminal side of the B chain/C peptide cleavage site in PI was cloned into the C‐terminal side of the K70‐R71 cleavage site in PG. In contrast to expectations, this substitution resulted in a nearly 50% decrease in PG cleavage by PCSK1. Equally surprising was that this substitution had almost no effect on the efficiency of PCSK2‐mediated cleavage of the altered site, despite the fact that in the original context of the PI substrate, this sequence is recognized much less efficiently by PCSK2 than by PCSK1. Taken together, these results suggest that three‐dimensional features (secondary or tertiary structure) play a significant role in the cleavage site specificity exhibited by both PCSK1 and PCSK2.

Distinct oxidative cleavage and modification of bovine [Cu– Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule

We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu–Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction.