Anthrax Toxin: Model System for Studying Protein Translocation.

Dedicated translocase channels are nanomachines that often, but not always, unfold and translocate proteins through narrow pores across the membrane. Generally, these molecular machines utilize external sources of free energy to drive these reactions, since folded proteins are thermodynamically stable, and once unfolded they contain immense diffusive configurational entropy. To catalyze unfolding and translocate the unfolded state at appreciable timescales, translocase channels often utilize analogous peptide-clamp active sites. Here we describe how anthrax toxin has been used as a biophysical model system to study protein translocation. The tripartite bacterial toxin is composed of an oligomeric translocase channel, protective antigen (PA), and two enzymes, edema factor (EF) and lethal factor (LF), which are translocated by PA into mammalian host cells. Unfolding and translocation are powered by the endosomal proton gradient and are catalyzed by three peptide-clamp sites in the PA channel: the α clamp, the ϕ clamp, and the charge clamp. These clamp sites interact nonspecifically with the chemically complex translocating chain, serve to minimize unfolded state configurational entropy, and work cooperatively to promote translocation. Two models of proton gradient driven translocation have been proposed: (i) an extended-chain Brownian ratchet mechanism and (ii) a proton-driven helix-compression mechanism. These models are not mutually exclusive; instead the extended-chain Brownian ratchet likely operates on ß-sheet sequences and the helix-compression mechanism likely operates on α-helical sequences. Finally, we compare and contrast anthrax toxin with other related and unrelated translocase channels.

Identification of a capsular polysaccharide from Enterococcus faecium U0317 using a targeted approach to discover immunogenic carbohydrates for vaccine development.

Enterococcus faecium, a gram-positive opportunistic pathogen, has become a major concern for nosocomial infections due to its resistance to several antibiotics, including vancomycin. Finding novel alternatives for treatment prevention, such as vaccines, is therefore crucial. In this study, we used various techniques to discover a novel capsular polysaccharide. Firstly, we identified an encapsulated E. faecium strain by evaluating the opsonophagocytic activity of fifteen strains with antibodies targeting the well-known lipoteichoic acid antigen. This activity was attributed to an unknown polysaccharide. We then prepared a crude cell wall glycopolymer and fractionated it, guided by immunodot-blot analysis. The most immunoreactive fractions were used for opsonophagocytic inhibition assays. The fraction containing the inhibitory polysaccharide underwent structural characterization using NMR and chemical analyses. The elucidated structure presents a branched repeating unit, with the linear part being: →)-ß-d-Gal-(1 â†’ 4)-ß-d-Glc-(1 â†’ 4)-ß-d-Gal-(1 â†’ 4)-ß-d-GlcNAc-(1→, further decorated with a terminal α-d-Glc and a d-phosphoglycerol moiety, attached to O-2 and O-3 of the 4-linked Gal unit, respectively. This polysaccharide was conjugated to BSA and the synthetic glycoprotein used to immunize mice. The resulting sera exhibited good opsonic activity, suggesting its potential as a vaccine antigen. In conclusion, our effector-function-based approach successfully identified an immunogenic capsular polysaccharide with promising applications in immunotherapy.

The polymer and materials science of the bacterial fimbriae Caf1.

Fimbriae are long filamentous polymeric protein structures located upon the surface of bacteria. Often implicated in pathogenicity, the biosynthesis and function of fimbriae has been a productive topic of study for many decades. Evolutionary pressures have ensured that fimbriae possess unique structural and mechanical properties which are advantageous to bacteria. These properties are also difficult to engineer with well-known synthetic and natural fibres, and this has raised an intriguing question: can we exploit the unique properties of bacterial fimbriae in useful ways? Initial work has set out to explore this question by using Capsular antigen fragment 1 (Caf1), a fimbriae expressed naturally by Yersina pestis. These fibres have evolved to 'shield' the bacterium from the immune system of an infected host, and thus are rather bioinert in nature. Caf1 is, however, very amenable to structural mutagenesis which allows the incorporation of useful bioactive functions and the modulation of the fibre's mechanical properties. Its high-yielding recombinant synthesis also ensures plentiful quantities of polymer are available to drive development. These advantageous features make Caf1 an archetype for the development of new polymers and materials based upon bacterial fimbriae. Here, we cover recent advances in this new field, and look to future possibilities of this promising biopolymer.

Predictive profiling of gram-negative antibiotics in CagA oncoprotein inactivation: a molecular dynamics simulation approach.

Gastric cancer (GC) is the fifth most prevalent form of cancer worldwide. CagA - positive Helicobacter pylori infects more than 60% of the human population. Moreover, chronic infection of CagA-positive H. pylori can directly affect GC incidence. In the current study, we have repurposed FDA-approved antibiotics that are viable alternatives to current regimens and can potentially be used as combination therapy against the CagA of H. pylori. The 100 FDA-approved gram negative antibiotics were screened against CagA protein using the AutoDock 4.2 tool. Further, top nine compounds were selected based on higher binding affinity with CagA. The trajectory analysis of MD simulations reflected that binding of these drugs with CagA stabilizes the system. Nonetheless, atomic density map and principal component analysis also support the notion of stable binding of antibiotics to the protein. The residues ASP96, GLN100, PRO184, and THR185 of compound cefpiramide, doxycycline, delafloxacin, metacycline, oxytetracycline, and ertapenem were involved in the binding with CagA protein. These residues are crucial for the CagA that aids in entry or pathogenesis of the bacterium. The screened FDA-approved antibiotics have a potential druggability to inhibit CagA and reduce the progression of H. pylori borne diseases.

The designation, synthesis, and affinity determination of affinity peptide for anthrax protective antigen.

Detection of anthrax protective antigen is an effective way to diagnose anthracnose, and it plays an important part in the treatment of anthracnose. Affinity peptides, as a miniature biological recognition element, can quickly and effectively detect anthrax protective antigens. Based on computer-aided design technology (CAD), we have herein developed an affinity peptide design strategy for the detection of anthrax protective antigens. Firstly, six high-value mutation sites were determined based on the molecular docking between the template peptide and the receptor, and then the multi-site mutation of amino acids was carried out in order to establish a virtual peptide library. The library was selected by using molecular dynamics simulation and the best designed affinity peptide (code: P24) was found. The theoretical affinity with P24 peptide has increased by 19.8% compared with template peptide. Finally, the affinity with P24 peptide was measured by SPR technology to reach the nanomole level, which verified the effectiveness of the design strategy. The newly designed affinity peptide is expected to be used in the diagnosis of anthracnose.

A conserved 3D pattern in a Streptococcus pyogenes M protein immunogen elicits M-type crossreactivity.

Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220 M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens because of type specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was shown to elicit M-type crossreactivity. The basis for this crossreactivity is unknown but may be due in part to antibody recognition of a 3D pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit crossreactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S. pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum-recognized M proteins displayed natively on the strep A surface and promoted the opsonophagocytic killing of strep A strains expressing these M proteins. As C4BP binding is a conserved virulence trait of strep A, we propose that targeting the 3D pattern may prove advantageous in vaccine design.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

A designed peptide-based vaccine to combat Brucella melitensis, B. suis and B. abortus: Harnessing an epitope mapping and immunoinformatics approach.

Vaccines against Brucella abortus, B. melitensis and B. suis have been based on weakened or killed bacteria, however there is no recombinant vaccine for disease prevention or therapy. This study attempted to predict IFN-γ epitopes, T cell cytotoxicity, and T lymphocytes in order to produce a multiepitope vaccine based on BtpA, Omp16, Omp28, virB10, Omp25, and Omp31 antigens against B. melitensis, B. abortus, and B. suis. AAY, GPGPG, and EAAAK peptides were used as epitope linkers, while the PADRE sequence was used as a Toll-like receptor 2 (TLR2) and TLR4 agonist. The final construct included 389 amino acids, and was a soluble protein with a molecular weight of 41.3 kDa, and nonallergenic and antigenic properties. Based on molecular docking studies, molecular dynamics simulations such as Gyration, RMSF, and RMSD, as well as tertiary structure validation methods, the modeled protein had a stable structure capable of interacting with TLR2/4. As a result, this novel vaccine may stimulate immune responses in B and T cells, and could prevent infection by B. suis, B. abortus, and B. melitensis.

Structure and role of the linker domain of the iron surface-determinant protein IsdH in heme transportation in Staphylococcus aureus.

Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAr-iron Transporter (NEAT) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route toward IsdH do not play a critical role in the transfer rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.

Directed Inter-domain Motions Enable the IsdH Staphylococcus aureus Receptor to Rapidly Extract Heme from Human Hemoglobin.

Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tri-domain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globin's heme pocket. Using molecular simulations coupled with Markov modeling, along with stopped-flow experiments to quantitatively measure heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hb's pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

Structure of the Anthrax Protective Antigen D425A Dominant Negative Mutant Reveals a Stalled Intermediate State of Pore Maturation.

The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of ß-barrel pore-forming toxins. The protective antigen (PA) component forms an oligomeric prepore that assembles on the host cell surface and serves as a scaffold for binding of lethal and edema factors. Following endocytosis, the acidic environment of the late endosome triggers a pH-induced conformational rearrangement to promote maturation of the PA prepore to a functional, membrane spanning pore that facilitates delivery of lethal and edema factors to the cytosol of the infected host. Here, we show that the dominant-negative D425A mutant of PA stalls anthrax pore maturation in an intermediate state at acidic pH. Our 2.7 Å cryo-EM structure of the intermediate state reveals structural rearrangements that involve constriction of the oligomeric pore combined with an intramolecular dissociation of the pore-forming module. In addition to defining the early stages of anthrax pore maturation, the structure identifies asymmetric conformational changes in the oligomeric pore that are influenced by the precise configuration of adjacent protomers.

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

Lyme Disease, Borrelia burgdorferi, and Lipid Immunogens.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

The Mycobacterium tuberculosis PE_PGRS Protein Family Acts as an Immunological Decoy to Subvert Host Immune Response.

Mycobacterium tuberculosis (M.tb) is a successful pathogen that can reside within the alveolar macrophages of the host and can survive in a latent stage. The pathogen has evolved and developed multiple strategies to resist the host immune responses. M.tb escapes from host macrophage through evasion or subversion of immune effector functions. M.tb genome codes for PE/PPE/PE_PGRS proteins, which are intrinsically disordered, redundant and antigenic in nature. These proteins perform multiple functions that intensify the virulence competence of M.tb majorly by modulating immune responses, thereby affecting immune mediated clearance of the pathogen. The highly repetitive, redundant and antigenic nature of PE/PPE/PE_PGRS proteins provide a critical edge over other M.tb proteins in terms of imparting a higher level of virulence and also as a decoy molecule that masks the effect of effector molecules, thereby modulating immuno-surveillance. An understanding of how these proteins subvert the host immunological machinery may add to the current knowledge about M.tb virulence and pathogenesis. This can help in redirecting our strategies for tackling M.tb infections.

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry.

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics.

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Binding of the kringle-2 domain of human plasminogen to streptococcal PAM-type M-protein causes dissociation of PAM dimers.

The direct binding of human plasminogen (hPg), via its kringle-2 domain (K2hPg ), to streptococcal M-protein (PAM), largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2 ), with one K2hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2hPg /PAMNS88.2 interaction. We show that apo-PAMNS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C-25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg /PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg /PAMNS88.2  monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2  monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg ) showing that PAM is a functional monomer at 37°C.

An electrochemical aptasensor for Mycobacterium tuberculosis ESAT-6 antigen detection using bimetallic organic framework.

A label-free electrochemical aptasensor is reported for sensitive detection of the 6-kDa early secreted antigenic target (ESAT-6). For the first time, the bimetallic organic framework (b-MOF) of Zr-MOF-on-Ce-MOF was decorated with nitrogen-doped graphene (NG) and applied as the matrix for electroactive toluidine blue (Tb) to form the NG@Zr-MOF-on-Ce-MOF@Tb nanohybrid. The prepared nanohybrid with excellent hydrophilicity, dispersibility, and large specific surface exhibited significant electrochemical response. This nanohybrid could be directly used for anchoring ESAT-6 binding aptamers (EBA) through the interaction between the 5'-phosphate group (PO43-) of EBA and Zr4+ of Zr-MOF. The signal response before and after incubating the ESAT-6 antigen has been evaluated by cyclic voltammetry at a scan rate of 100 mV s-1 from - 0.7 to 0.3 V (vs. SCE). Under optimal conditions, the proposed aptasensor displayed a wide linear range from 100 fg mL-1 to 10 ng mL-1 with a limit of detection (LOD) of 12 fg mL-1. The developed method showed good reproducibility with a relative standard deviation (RSD) of 2.27%. The aptasensor showed favorable results in the analysis of the real samples. With these merits, the aptasensor has exceptional potential as a diagnostic tool for tuberculosis in clinical practice.

Bottom-up fabrication of a proteasome-nanopore that unravels and processes single proteins.

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise ß-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides.