A Pyroglutamate Aminopeptidase 1 Responsive Fluorescence Imaging Probe for Real-Time Rapid Differentiation between Thyroiditis and Thyroid Cancer.

Current diagnostic methods for thyroid diseases, including blood tests, ultrasound, and biopsy, always have difficulty diagnosing thyroiditis accurately, occasionally mistaking it for thyroid cancer. To address this clinical challenge, we developed Ox-PGP1, a novel fluorescent probe realizing rapid, noninvasive, and real-time diagnostic techniques. This is the first imaging tool capable of noninvasively distinguishing between thyroiditis and thyroid cancer. Ox-PGP1 was introduced as a fluorescent probe custom-built for the specific detection and quantification of pyroglutamate aminopeptidase 1 (PGP-1), a known pivotal biomarker of inflammation. Ox-PGP1 overcame the disadvantages of traditional enzyme-responsive fluorescent probes that relied on the intramolecular charge transfer (ICT) mechanism, including the issue of high background fluorescence, while offering exceptional photostability under laser irradiation. The spectral properties of Ox-PGP1 were meticulously optimized to enhance its biocompatibility. Furthermore, the low limit of detection (LOD) of Ox-PGP1 was determined to be 0.09 µg/mL, which demonstrated its remarkable sensitivity and precision. Both cellular and in vivo experiments validated the capacity of Ox-PGP1 for accurate differentiation between normal, inflammatory, and cancerous thyroid cells. Furthermore, Ox-PGP1 showed the potential to rapidly and sensitively differentiate between autoimmune thyroiditis and anaplastic thyroid carcinoma in a mouse model, achieving results in just 5 min. The successful design and application of Ox-PGP1 represent a substantial advancement in technology over traditional diagnostic approaches, potentially enabling earlier interventions for thyroid diseases.

Pyroglutamate Aminopeptidase I Promotes Hepatocellular Carcinoma via IL-6/STAT3 Activation as Revealed by a Specific Biosensor.

As a global health challenge, hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. Targeting inflammation, particularly inflammatory factors, is regarded as an important strategy for HCC diagnosis and treatment. Pyroglutamic aminopeptidase I (PGP-I), a common exopeptidase, was recently identified as a novel inflammatory cytokine in cells. However, whether PGP-I is involved in HCC development and can be regarded as a biomarker remains unclear. To address this issue, endogenous PGP-I was imaged in live cells and in vivo, and the related biochemical and pathological processes were analyzed accordingly with a newly developed fluorogenic PGP-I biosensor. Bioimaging with the specific biosensor demonstrated the aberrant expression of PGP-I in HCC cell lines and tumor-bearing nude mice. Moreover, overexpression of PGP-I in HCC cells promoted tumor progression, whereas knockdown of PGP-I significantly suppressed tumor cell growth and migration. The activity of PGP-I was further identified to be highly related to the phosphorylation of STAT3, which could be impeded by the natural product parthenolide. Collectively, these findings suggest that PGP-I, which can promote hepatocellular tumor progression through the classical inflammation-/tumor-related IL-6/STAT3 pathway, may serve as a potential HCC biomarker and therapeutic target.

A bioluminescent probe for in vivo imaging of pyroglutamate aminopeptidase in a mouse model of inflammation.

Pyroglutamate aminopeptidase (PGP) specifically cleaves the peptide bond of pyroglutamic acid linked to the N-terminal end of a polypeptide or protein. Previous studies showed that PGP was associated with several physiological processes and diseases especially those involving inflammation. Utilizing a 'caging' strategy, we designed and synthesized a bioluminescence probe (PBL) with a limit-of-detection of 3.7 * 10-4 mU/mL. In vivo imaging in a mouse model of inflammatory liver disease revealed that the probe has excellent sensitivity and selectivity and provides a powerful tool for studying the physiological and pathological processes involving PGP.

Neoadjuvant chemotherapy modifies serum pyrrolidone carboxypeptidase specific activity in women with breast cancer and influences circulating levels of GnRH and gonadotropins.

PURPOSE: Functional studies have demonstrated that gonadotropin-releasing hormone (GnRH) regulates cell proliferation, apoptosis, and tissue remodeling. GnRH is metabolized by the proteolytic regulatory enzyme pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3), which is an omega peptidase widely distributed in fluids and tissues. We previously reported a decrease in both rat and human Pcp activity in breast cancer, suggesting that GnRH may be an important local hormonal factor in the pathogenesis of breast cancer. Recently, we have described that postmenopausal women with breast cancer show lower levels of serum Pcp activity than control postmenopausal women. To determine the effect of neoadjuvant chemotherapy (NACT) on serum Pcp specific activity and circulating levels of GnRH, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and steroid hormones 17-ß-estradiol and progesterone in pre- and postmenopausal women diagnosed with infiltrating ductal carcinoma. METHODS: Serum Pcp activity was measured fluorometrically using pyroglutamyl-ß-naphthylamide. Circulating GnRH levels were dosed using a commercial RIA kit. Circulating LH and FSH levels were measured by enzyme immunoassays. Levels of steroid hormones were measured in serum samples by dissociation-enhanced lanthanide fluorescence immunoassay. RESULTS AND CONCLUSION: Our results show the effect of NACT on the hypothalamic-pituitary axis, with the consequent alteration of circulating gonadotropins in premenopausal women with breast cancer. However, the results obtained in postmenopausal women with breast cancer treated with NACT, that is, the significant decrease in the concentration of GnRH and FSH compared to control postmenopausal women, differ from those obtained for premenopausal women. The only difference between pre- and postmenopausal women is their hormonal profile at the beginning of the study, that is, the presence of menopause and the consequent alteration of the hypothalamic-pituitary-gonadal axis.

Isolation of an anti-entomopathogenic fungal protein secreted from Pseudomonas aeruginosa BGf-2: An intestinal bacteriam of Blattella germanica (L.).

A bacterial strain (BGf-2) with anti-Beauveria bassiana activity was obtained from the feces of Blattella germanica (L.) and identified as Pseudomonas aeruginosa based on biochemical tests and 16S rRNA sequence analysis. An antifungal protein (A0A0H2ZK06) was purified with Sephadex G-100 column and DEAE-sepharose Fast Flowanion exchange from sterile BGf-2 fermentation liquid. Based on MALDI-TOF MS analysis and protein model building, A0A0H2ZK06 showed homology with Pyrrolidone carboxyl peptidases (pcps). Fermentation liquid and antifungal proteins not only reduced the B. bassiana conidial germination rate but also inhibited hyphal growth. A per os test showed that the mortality of cockroaches decreased after treatment with BGf-2 suspension compared with control. We hypothesized that gut microbes with antifungal activity might play an important role in protect cockroaches from pathogenic fungi.

Altered Activity and Expression of Cytosolic Peptidases in Colorectal Cancer.

BACKGROUND AND OBJECTIVE: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. METHODS: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). RESULTS: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall survival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. CONCLUSIONS: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.

Crystal structure analysis of pyrrolidone carboxyl peptidase from Thermus thermophilus.

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/ß/α architecture in which the central ß-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel ß-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.

Circ-PGPEP1 augments renal cell carcinoma proliferation, Warburg effect, and distant metastasis.

Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including renal cell carcinoma (RCC). This study probed the molecular mechanism of circPGPEP1 regulating RCC proliferation, Warburg effect, and distant metastasis by targeting the miR-378a-3p/JPT1 axis. Here identified higher circPGPEP1 expression in RCC tissues and cells by RT-qPCR, and high levels of circPGPEP1 were positively correlated with high histological grade and distant metastasis in RCC patients. Furthermore, patients with high levels of circPGPEP1 had a worse survival prognosis. Functional assays presented that knockdown of circPGPEP1 inhibited RCC proliferation, invasion, migration, EMT, and Warburg effect. Dual-luciferase reporter assay, RNA immunoprecipitation, nucleoplasmic RNA isolation, and functional rescue experiments confirmed that circPGPEP1 induced JPT1 expression by sponging miR-378a-3p, thereby promoting RCC malignant phenotype. Xenograft assays and metastasis models further demonstrated that down-regulation of circPGPEP1 effectively inhibited tumor growth and distant metastasis of RCC. Taken together, circPGPEP1, a prognostic circRNA in RCC, acts through the miR-378a-3p/JPT1 axis to regulate RCC progression.

Structural characterization of a trapped folding intermediate of pyrrolidone carboxyl peptidase from a hyperthermophile.

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central ß-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.

N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies.

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.

Two-photon fluorogenic probe for visualizing PGP-1 activity in inflammatory tissues and serum from patients.

A PGP-1-specific one/two-photon fluorogenic probe (BH1), capable of high sensitivity, super selectivity, and visual imaging of endogenous PGP-1 activity from live mammalian cells and serum/skin tissues from patients by using one/two-photon fluorescence microscopy (O/TPFM).

A ratiometric fluorescent sensor for rapid detection of the pyroglutamate aminopeptidase-1 in mouse tumors.

Pyroglutamate aminopeptidase-1 (PGP-1) is an important enzyme that plays an indispensable role in the process of inflammation. Up to now, few reports have been reported on the detection of PGP-1 activity in vivo and in vitro, and there are no reports on ratiometric detection. Here, the first red-emitting ratiometric fluorescent sensor (DP-1) for the specific detection of PGP-1 both in vivo and in vitro was designed and synthesized by using DCD-NH2 as the luminescent parent and pyroglutamate as a recognition group. After interacting with PGP-1, the amide bond is hydrolyzed by the enzyme and the color of the solution changes from yellow (λabs = 420 nm) to red (λabs = 520 nm), accompanied by obvious fluorescence emission wavelength change (from ∼564 nm to ∼616 nm). The probe has high specificity and sensitivity towards PGP-1 in about 10 min, and the DL is as low as 0.25 ng mL-1. Interestingly, under the stimulation of Freund's incomplete adjuvant and lipopolysaccharide, the imaging of DP-1 in HepG2 and RAW264 cells shows that the expression of PGP-1 is associated with inflammation. What's more, for the first, the imaging of a mouse tumor model confirms that the enzyme is closely related to the occurrence of some inflammation and tumor diseases. These results indicate that DP-1 can be used as an effective tool for real-time monitoring of PGP-1 levels both in vivo and in vitro and the study of inflammatory tumor pathology.

The Role of High Fat Diets and Liver Peptidase Activity in the Development of Obesity and Insulin Resistance in Wistar Rats.

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of ß-cell usefulness (% ß-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism.

Crystal structures of pyrrolidone-carboxylate peptidase I from Deinococcus radiodurans reveal the mechanism of L-pyroglutamate recognition.

Pyrrolidone-carboxylate peptidase (PCP) catalyzes the removal of an unusual amino acid, L-pyroglutamate (pG), from the N-termini of peptides and proteins. It has implications in the functional regulation of different peptides in both prokaryotes and eukaryotes. However, the pG-recognition mechanism of the PCP enzyme remains largely unknown. Here, crystal structures of PCP I from Deinococcus radiodurans (PCPdr) are reported in pG-free and pG-bound forms at resolutions of 1.73 and 1.55 Å, respectively. Four protomers in PCPdr form a tetrameric structure. The residues responsible for recognizing the pG residue are mostly contributed by a flexible loop (loop A) that is present near the active site. These residues are conserved in all known PCPs I, including those from mammals. Phe9 and Phe12 of loop A form stacking interactions with the pyrrolidone ring of pG, while Asn18 forms a hydrogen bond to OE of pG. The main chain of a nonconserved residue, Leu71, forms two hydrogen bonds to NH and OE of pG. Thus, pG is recognized in the S1 substrate subsite of the enzyme by both van der Waals and polar interactions, which provide specificity for the pG residue of the peptide. In contrast to previously reported PCP I structures, the PCPdr tetramer is in a closed conformation with an inaccessible active site. The structures show that the active site can be accessed by the substrates via disordering of loop A. This disordering could also prevent product inhibition by releasing the bound pG product from the S1 subsite, thus allowing the enzyme to engage a fresh substrate.

Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

The confirmation of the denatured structure of pyrrolidone carboxyl peptidase under nondenaturing conditions: difference in helix propensity of two synthetic peptides with single amino acid substitution.

In the denatured state (D(1) state) of cystein-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus, a hyperthermophile under nondenaturing conditions, a fairly stable alpha-helix (alpha6-helix) has been determined from H/D exchange-NMR experiments. On the other hand, the alpha6-helix region of the proline-mutant at position 199 (A199P) was unstructured in the D(1) state unlike that of the wild-type PCP-0SH, although the folded conformations of both proteins were almost identical to each other. This finding has been deduced from the information regarding the remaining amide hydrogens in the HSQC spectra after H/D exchanges in the D(1) state. To confirm this inference, we examined the helical propensities of two synthetic peptides from their NMR structural analysis in the presence of trifluoroethanol (TFE). One is an 18-residue peptide called the wild-type H6-peptide corresponding to the alpha6-helix (from Ser188 to Glu205) of the wild-type PCP-0SH, and the other is the mutant H6-peptide corresponding to the alpha6-helix region of A199P. The NOE-contact information obtained from the 2D-(1)H-NOESY spectra measured for both peptides in the presence of 30% TFE clearly demonstrated that the wild-type H6-peptide had a high helical propensity, but the mutant H6-peptide was almost totally unstructured. The TFE-induced helical propensities for these peptide fragments confirmed the conclusions deduced from the H/D exchange data measured in the D(1) states of two proteins.

Microbiological characteristics, presumptive identification, and antibiotic susceptibilities of Staphylococcus lugdunensis.

This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was detected in 27 and 5% of isolates, respectively.

Chronic ethanol intake modifies pyrrolidon carboxypeptidase activity in mouse frontal cortex synaptosomes under resting and K+ -stimulated conditions: role of calcium.

Pyrrolidon carboxypeptidase (Pcp) is an omega peptidase that removes pyroglutamyl N-terminal residues of peptides such as thyrotrophin-releasing hormone (TRH), which is one of the neuropeptides that has been localized into many areas of the brain and acts as an endogenous neuromodulator of several parameters related to ethanol (EtOH) consumption. In this study, we analysed the effects of chronic EtOH intake on Pcp activity on mouse frontal cortex synaptosomes and their corresponding supernatant under basal and K+ -stimulated conditions, in presence and absence of calcium (Ca2+) to know the regulation of Pcp on TRH. In basal conditions, chronic EtOH intake significantly decreased synaptosomes Pcp activity but only in absence of Ca2+. However, supernatant Pcp activity is also decreased in presence and absence of calcium. Under K+-stimulated conditions, chronic EtOH intake decreased synaptosomes Pcp activity but only in absence of Ca2+, whereas supernatant Pcp activity was significantly decreased only in presence of Ca2+. The general inhibitory effect of chronic EtOH intake on Pcp activity suggests an inhibition of TRH metabolism and an enhancement of TRH neurotransmitter/neuromodulator functions, which could be related to putative processes of tolerance to EtOH in which TRH has been involved. Our data may also indicate that active peptides and their degrading peptidases are released together to the synaptic cleft to regulate the neurotransmitter/neuromodulator functions of these peptides, through a Ca2+ -dependent mechanism.

Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues.

To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 degrees C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 degrees C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.