Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4⁺ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii⁻ cells may present peptides not presented by Ii⁺ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii⁺ cells, but will not be tolerant to novel peptides presented by Ii⁻ cells, we generated MHC II vaccines to activate cancer patients'' T cells. The vaccines are Ii⁻ tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii⁺ and Ii⁻ MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii⁻ cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii⁻ and Ii⁺ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii⁻ cells are not presented by Ii⁺ cells, and are derived from source proteins not used by Ii⁺ cells. Peptides from Ii⁻ cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7⁺ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II⁺ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.Cancer vaccines are a promising tool for cancer treatment and prevention because of their potential for inducing tumor-specific responses in conjunction with minimal toxicity for healthy cells. Cancer vaccines are based on the concept that tumor cells synthesize multiple peptides that are potential immunogens, and that with the appropriate vaccine protocol, these peptides will activate an efficacious antitumor response in the patient. Much effort has been invested in identifying and testing tumor-encoded peptides, particularly peptides presented by major histocompatibility complex (MHC)¹ class I, molecules capable of activating CD8⁺ T-cells that directly kill tumor cells (1, 2). Fewer studies have been devoted to identifying MHC class II-restricted peptides for the activation of tumor-reactive CD4⁺ T-cells despite compelling evidence that Type 1 CD4⁺ T helper cells facilitate the optimal activation of CD8⁺ T-cells and the generation of immune memory, which is likely to be essential for protection from metastatic disease.Activation of CD4⁺ T cells requires delivery of a costimulatory signal plus an antigen-specific signal consisting of peptide bound to an MHC II molecule. Most cells do not express MHC II or costimulatory molecules, so CD4⁺ T cells are typically activated by professional antigen presenting cells (APC), which endocytose exogenously synthesized antigen and process and present it in the context of their own MHC II molecules. This processing and presentation process requires Invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of APC-encoded endogenous peptides (3, 4). As a result, tumor-reactive CD4⁺ T cells are activated to tumor peptides generated by the antigen processing machinery of professional APC, rather than peptides generated by the tumor cells. Because of the potential discrepancy in peptide generation between professional APC and tumor cells, and the critical role of Ii in preventing the presentation of endogenous peptides, we have generated “MHC II cancer vaccines” that consist of Ii⁻ tumor cells transfected with syngeneic MHC class II and CD80 genes. We reasoned that MHC II⁺Ii⁻CD80⁺ tumor cells may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APC, and therefore may be highly immunogenic. Once activated, CD4⁺ T cells produce IFNγ and provide help to CD8⁺ T cells and do not need to react with native tumor cells. Therefore, the MHC II vaccines have the potential to activate CD4⁺ Th1 cells that facilitate antitumor immunity. In vitro (5) and in vivo (5–7) studies with mice support this conclusion. In vitro studies with human MHC II vaccines further demonstrate that the absence of Ii facilitates the activation of MHC II-restricted tumor-specific CD4⁺ type 1 T cells of HLA-DR-syngeneic healthy donors and cancer patients, and that the vaccines activate CD4⁺ T cells with a distinct repertoire of T cell receptors (8–12). A critical negative role for Ii is also supported by studies of human acute myelogenous leukemia (AML). High levels of class II-associated invariant chain peptide (CLIP), a degradation product of Ii, by leukemic blasts is associated with poor patient prognosis (13, 14), whereas down-modulation of CLIP on AML cells increases the activation of tumor-reactive human CD4⁺ T cells (14, 15).We have now used mass spectrometry to identify MHC II-restricted peptides from MHC II⁺Ii⁻ and MHC II⁺Ii⁺ human breast cancer cells to test the concept that the absence of Ii facilitates the presentation of unique immunogenic MHC II-restricted peptides. We report here that a subset of MHC II-restricted peptides from HLA-DR7⁺ breast cancer cells are unique to Ii⁻ cells and are derived from source proteins not used by Ii⁺ cells. Ii⁻ peptides have high binding affinity for HLA-DR7 and activate tumor-specific T-cells from the peripheral blood of healthy donors and breast cancer patients. This is the first study to compare the human tumor cell MHC II peptidome in the absence or presence of Ii and to demonstrate that MHC II⁺Ii⁻ tumor cells present novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.     

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.     

CytokineDB: a database collecting biological information

Cytokines are subdivided in 12 sub-families and are described as multi-functional molecules that play an important biological activity in host defense system against pathogens, in homeostasis, tissue repair, cell growth and development. CytokineDB is an annotated database that collects biological information regarding the cytokines family in human and will be periodically updated by including new biological information. This database is freely available online and can be accessed at the URL: http://www.cro-m.eu/CytokineDB/     

Seed Pro-Nutra Care: A tool for characterization of seed storage proteins and database of bioactive peptides having potential health benefits

Seed storage proteins, the major food proteins, possess unique physicochemical characteristics which determine their nutritional importance and influence their utilization by humans. Here, we describe a database driven tool named Seed Pro-Nutra Care which comprises a systematic compendium of seed storage proteins and their bioactive peptides influencing several vital organ systems for maintenance of health. Seed Pro-Nutra Careis an integrated resource on seed storage protein. This resource help in the (I) Characterization of proteins whether they belong to seed storage protein group or not. (II) Identification the bioactive peptides with their sequences using peptide name (III) Determination of physico chemical properties of seed storage proteins. (IV) Epitope identification and mapping (V) Allergenicity prediction and characterization. Seed Pro-Nutra Care is a compilation of data on bioactive peptides present in seed storage proteins from our own collections and other published and unpublished sources. The database provides an information resource of a variety of seed related biological information and its use for nutritional and biomedical application.

Availability

http://www.gbpuat-cbsh.ac.in/departments/bi/database/seed_pro_nutra_care/     

PDBsum extras: SARS‐CoV‐2 and AlphaFold models

The PDBsum web server provides structural analyses of the entries in the Protein Data Bank (PDB). Two recent additions are described here. The first is the detailed analysis of the SARS‐CoV‐2 virus protein structures in the PDB. These include the variants of concern, which are shown both on the sequences and 3D structures of the proteins. The second addition is the inclusion of the available AlphaFold models for human proteins. The pages allow a search of the protein against existing structures in the PDB via the Sequence Annotated by Structure (SAS) server, so one can easily compare the predicted model against experimentally determined structures. The server is freely accessible to all at http://www.ebi.ac.uk/pdbsum.     

Simulation of Major Histocompatibility Complex (MHC) structure and peptide loading into an MHC binding pocket with teachers’hands

Molecular understanding of three-dimensional (3D) peptide: MHC models require both basic knowledge of computational modeling and skilled visual perception, which are not possessed by all students. The present model aims to simulate MHC molecular structure with the hands and make a profound impression on the students.Key Words: MHC molecules, Simulation, Hand, Binding pocket, Physical model using handsCurrently, lecturers can use many instructional models to enhance teaching of molecular structures to undergraduate students. However, we sometimes find it necessary to present an immunological concept with a touchable model to create a more understandable and profound image than can be found in books. The realization of paratope-epitope interaction with both hands and an apple is one example of this simulation in immunology teaching (1). MHC class I and II structures and MHC peptide-loading compartments are considered to be fundamental concepts in immunology. Molecular understanding of 3D peptide: MHC models require both basic knowledge of computational modeling and skilled visual perception, which are not possessed by all audiences. However, no models are more accessible, efficient, and affordable than teachers’ hands. This simulation aims to simplify perception of MHC molecular structures and provide an enduring image of MHC-peptide interactions. Simulation of MHC class I structure and its peptide loading compartment To imitate MHC class I structure with the hands, bend the index, middle, and ring fingers toward the palm and then touch the thumb with the little finger as shown in Fig. 1. The three bent fingers and the connected thumb and little finger simulate the pair of parallel α-helices that form the peptide binding groove. One helix belongs to the α1 domain and the other to the α2 domain. The palm represents the beta-pleated sheet structure, which forms the base of the groove. The narrow groove between the bent and connected fingers represents the peptide-loading compartment. A flexible rod inserted into this groove can be imagined as an antigenic peptide located in the closed groove of the MHC class I molecule. The rod must bend in the middle to simulate the loaded peptide. Introduce your left hand to the students as an α-chain of MHC class I structure, which is encoded on chromosome 6 in humans. Then, introduce your watch as a β2-microglobulin chain that is non-covalently associated with the α-chain and encoded on chromosome 15 in humans. It should be emphasized that the polypeptide chains are encoded by genes on different chromosomes. The MHC class I α-chain is inserted into the cell membrane, indicated by the shirt sleeve in our simulation (2, 3). Open in a separate windowFig. 1Simulation of MHC class I structure and its peptide loading compartment (A). In B this simulation has been superimposed with two α-helices and a beta-pleated sheet of the α-chain from a 3D view of human class I MHC HLA-A2 (3PWJ). Simulation of MHC class II structure and its peptide loading compartment To demonstrate MHC class II structure, bend all the fingers of both hands toward the palms and then close both hands from the forearms as shown in Fig. 2. Try to hide your watch, which represented β2-microglobulin. The bent fingers and palm of the left hand represent the α-helix and β-pleated sheet of the α-chain, respectively (Fig. 2), and the same status could be imagined for the right hand as a β-chain of MHC class II. The open space between the bent fingers of both hands represents the peptide anchoring site, which is an open-ended groove in MHC class II. A flexible rod with overhanging portions projecting from both sides of groove inserted in this space simulates an antigenic peptide that has been located in an open-ended groove of the MHC class II molecule. MHC class II molecules bind peptides of 13-25 amino acids, which is considerably longer than MHC class I-binding peptides of 8-11 amino acids. The two chains of MHC class II molecules are inserted into the cell membrane, represented by the shirt sleeves in our simulation. In this simulation, the two chains of the MHC class II molecules are represented by the two hands, while the MHC class I molecule was modeled by a hand and a watch. This is a good time to remind the students that both chains of MHC class II molecules, in contrast to class I molecules, are encoded by the MHC gene cluster on chromosome 6 in humans, and that the two chains associate non-covalently (2, 3).Open in a separate windowFig. 2Simulation of MHC class II structure and its peptide loading compartment (A). This simulation has been superimposed with two α-helices and two beta-pleated sheets of α-chain and β-chain from the crystal structure of HLA-DR1 (3PDO) (B).Certainly, some MHC structural features are not completely duplicated with this model, but this visualization of a molecular structure makes a profound impression on the students.     

Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3-36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.     

Genetics of Lipid-Storage Management in Caenorhabditis elegans Embryos

Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)–treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L–like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease–causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms.     

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.     

Reversal of Salt Preference Is Directed by the Insulin/PI3K and Gq/PKC Signaling in Caenorhabditis elegans

Animals search for foods and decide their behaviors according to previous experience. Caenorhabditis elegans detects chemicals with a limited number of sensory neurons, allowing us to dissect roles of each neuron for innate and learned behaviors. C. elegans is attracted to salt after exposure to the salt (NaCl) with food. In contrast, it learns to avoid the salt after exposure to the salt without food. In salt-attraction behavior, it is known that the ASE taste sensory neurons (ASEL and ASER) play a major role. However, little is known about mechanisms for learned salt avoidance. Here, through dissecting contributions of ASE neurons for salt chemotaxis, we show that both ASEL and ASER generate salt chemotaxis plasticity. In ASER, we have previously shown that the insulin/PI 3-kinase signaling acts for starvation-induced salt chemotaxis plasticity. This study shows that the PI 3-kinase signaling promotes aversive drive of ASER but not of ASEL. Furthermore, the G_q signaling pathway composed of G_qα EGL-30, diacylglycerol, and nPKC (novel protein kinase C) TTX-4 promotes attractive drive of ASER but not of ASEL. A putative salt receptor GCY-22 guanylyl cyclase is required in ASER for both salt attraction and avoidance. Our results suggest that ASEL and ASER use distinct molecular mechanisms to regulate salt chemotaxis plasticity.ANIMALS show various behaviors in response to environmental cues and modulate behaviors according to previous experience. To understand neuronal plasticity underlying learning, it is important to dissect neurons and molecules for sensing environmental stimuli, storing memory, and executing learned behaviors.The nematode Caenorhabditis elegans has only 302 neurons and functions of sensory neurons are well characterized (White et al. 1986; Bargmann 2006). C. elegans is attracted to odorants sensed by the AWC olfactory neurons or to salts sensed by the ASE gustatory neurons (Bargmann and Horvitz 1991; Bargmann et al. 1993). The ASE neuron class consists of a bilaterally symmetrical pair, ASE-left (ASEL) and ASE-right (ASER), which sense different sets of ions including Na⁺ and Cl⁻, respectively (Pierce-Shimomura et al. 2001; Suzuki et al. 2008; Ortiz et al. 2009). ASEL is activated by an increase in salt concentration, whereas ASER is activated by a decrease in salt concentration (Suzuki et al. 2008). In the ASE gustatory neurons, a cyclic GMP (cGMP) signaling pathway mediates sensory transduction (Komatsu et al. 1996; Suzuki et al. 2008; Ortiz et al. 2009). ASEL and ASER express different sets of receptor-type guanylyl cyclases (gcys) (Ortiz et al. 2006). Of these, gcy-22, which is specifically expressed in ASER, is important for attraction to ASER-sensed ions such as Cl⁻ (Ortiz et al. 2009).Preference for salts changes according to previous experience (known as gustatory plasticity or salt chemotaxis learning) (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). When worms are grown on a medium that contains sodium chloride (NaCl) and food (Escherichia coli), they show attraction to NaCl by using ASE neurons (Bargmann and Horvitz 1991; Suzuki et al. 2008). In contrast, after exposure to the salt under starvation conditions, they show reduced attraction to or even avoid the salt (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). In C. elegans, it was proposed that preference for a sensory cue is defined by the sensory neuron that detects the cue (Troemel et al. 1997). ASE neurons play a major role for salt attraction (Bargmann and Horvitz 1991; Suzuki et al. 2008; Ortiz et al. 2009). However, little is known about sensory neurons that drive the learned salt avoidance; it remains unclear whether ASE neurons act as salt receptors for the learned avoidance.We have previously shown that an insulin/PI 3-kinase signaling pathway is essential for salt chemotaxis learning (Tomioka et al. 2006). In C. elegans, the insulin-like signaling is composed of daf-2, age-1, and akt-1, which encode homologs of insulin receptor, PI 3-kinase, and protein kinase B, respectively (Morris et al. 1996; Kimura et al. 1997; Paradis and Ruvkun 1998). Mutants of daf-2, age-1, and akt-1 show attraction to salt even after starvation/NaCl conditioning (Tomioka et al. 2006).daf-18 encodes a homolog of phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates phosphatidylinositol (3,4,5)-triphosphate and counteracts the insulin/PI 3-kinase signaling (Ogg and Ruvkun 1998; Gil et al. 1999; Mihaylova et al. 1999; Rouault et al. 1999; Solari et al. 2005). Mutants of daf-18, in which the PI 3-kinase signaling is activated, show reduced attraction to NaCl even without conditioning. Since the insulin/PI 3-kinase signaling acts in ASER, we proposed that the insulin/PI 3-kinase signaling attenuates the attractive drive of ASER (Tomioka et al. 2006).In C. elegans, diacylglycerol (DAG) regulates functions of motor neurons and sensory neurons. egl-30, which encodes the α-subunit of heterotrimeric G-protein G_q, facilitates production of DAG and enhances locomotory movements (Brundage et al. 1996; Lackner et al. 1999). In the AWC olfactory neurons, a novel protein kinase C-ɛ/η (nPKC-ɛ/η) ortholog TTX-4 (also known as PKC-1), which is one of DAG targets, plays an essential role in attraction behavior to AWC-sensed odors (Okochi et al. 2005; Tsunozaki et al. 2008). GOA-1 G_oα regulates olfactory adaptation by antagonizing G_qα–DAG signaling (Matsuki et al. 2006).This study investigated the involvement of the ASE taste receptor neurons in the starvation-induced salt avoidance. We show that both ASEL and ASER contribute to salt chemotaxis learning. Activation of the PI 3-kinase signaling and the G_q/DAG/PKC signaling acted antagonistically in reversal of ASER function, whereas these signaling pathways did not have prominent effects on ASEL function. In ASER, GCY-22 was required for both salt attraction and avoidance. These results suggest that ASE neurons are important for bidirectional chemotaxis and also suggest that distinct molecular mechanisms regulate functions of ASEL and ASER in salt chemotaxis learning.     

Decreasing Trend of Overlapping Multilocus Sequence Types between Human and Chicken Campylobacter jejuni Isolates over a Decade in Finland

We describe the long-term multilocus sequence typing (MLST) analysis of the population structure and dynamics of 454 Finnish human Campylobacter jejuni isolates, as well as 208 chicken isolates, collected during the mid-1990s to 2007. The sequence type clonal complexes (ST CC) ST-45 CC, ST-21 CC, and ST-677 CC were the most common ones found among all isolates, and they covered 73.9% of all isolates. The ST-283 CC also was found frequently among chicken isolates (8.2%). The predominant STs among all isolates were ST-45, ST-50, and ST-677. ST-137 and ST-230 were common among human isolates, and ST-267 was found more frequently among chicken isolates than human isolates. The ST-45 CC was significantly associated with chicken isolates (P < 0.01), whereas the ST-21 CC was associated with human isolates (P < 0.001). The ST-677 CC was not associated with any host (P = 0.5), and an opposite temporary trend of this complex was seen among chicken and human isolates, with an increase in the former and a decrease in the latter during the study period. Furthermore, the ST-22 and ST-48 CCs were significantly associated with human isolates (P < 0.01), but neither of the CCs was found in chicken isolates. The annual overlap between STs from human and chicken isolates decreased from 76% at the beginning of the study to 58% at the end. Our results suggest that the importance of chicken as a reservoir for strains associated with human infections has declined despite the consumption of domestic chicken meat increasing during the follow-up period by 83%.Campylobacter jejuni is the most common bacterial cause of gastroenteritis worldwide. In Finland, registered human Campylobacter infections have increased from 2,629 cases in 1996 to 4,107 cases in 2007 (http://www3.ktl.fi/stat/). Of the 4,107 cases in 2007, 45% were registered in the Helsinki-Uusimaa region, where the annual incidence was highest (122/100,000; the national average is 78/100,000) as well. Most of the cases were associated with traveling to other countries. In 2007, approximately 57% of the patients had traveled outside of Finland prior to their illness, 19% had not traveled abroad, and for 24% information was unavailable (http://www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf).In Finland, most of the Campylobacter infections are sporadic and appear during the summer months (http://www.efsa.europa.eu/en/scdocs/doc/130r.pdf). Between 1996 and 2007, approximately 45 to 55% of all registered infections were reported between June and September (http://www3.ktl.fi/stat/). In contrast to sporadic Campylobacter infections, outbreaks of campylobacteriosis are uncommon, usually occurring in spring or autumn, and are associated with drinking contaminated water (36).Epidemiological studies performed in many countries indicate that handling or eating chicken meat is an important risk factor for the acquisition of campylobacteriosis (32, 46). A recent Finnish case-control study (39) identified swimming in lakes and rivers and drinking water from private wells as additional risk factors for acquiring the illness from domestic sources during the summer months. Contact with pets (19) and farm animals (21) and the consumption of raw milk (35) also have been identified as risk factors for Campylobacter infections.The annual consumption of chicken meat in Finland increased by 83% (from 53 million to 95 million kg) from 1997 to 2007 but remained stable, at around 83.5 million kg, between 2002 and 2006. However, from 2006 to 2007, the consumption of chicken meat increased by a further 8% (from 88 million to 95 million kg). Most of the chicken meat consumed in Finland comes from domestic production (https://portal.mtt.fi/portal/page/portal/mtt/mtt/julkaisut/suomenmaatalousjamaaseutuelinkeinot/jul108_SM2008.pdf). Finnish chicken flocks have been monitored for Campylobacter spp. according to the regulations of the European Union since 2004 (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:325:0031:0040:EN:PDF). Seasonality similar to that observed in human infections can be found in the prevalence of Campylobacter-positive chicken slaughter batches, i.e., 7.7% of all chicken slaughter batches were Campylobacter positive from June to October in 2007; however, during the rest of the year no positive chicken flocks were detected (http://www.efsa.europa.eu/en/scdocs/doc/130r.pdf).To assess the relevance of a particular source and potential routes of transmission from animals to humans (6), the overlap between genotypes of Campylobacter isolates from patients and potential sources of infection has been studied using molecular typing techniques such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Of the two typing methods, PFGE is more discriminatory and therefore is considered more suitable for short-term epidemiological investigations and for the determination of the source of infection in outbreak situations (24, 26, 28).In our previous studies, we have shown that during a seasonal peak, overlapping serotypes and PFGE genotypes exist between samples from patients suffering from campylobacteriosis and fecal samples from chicken (13, 17). Several other studies also have reported a 34 to 60% overlap between serotypes, genotypes, and/or sequence types (STs) of patient and poultry isolates using various typing techniques (20, 31, 37, 42).Due to the high diversity of types and the lack of standardized nomenclature, PFGE is not a useful technique in long-term epidemiological studies. Unlike PFGE, MLST has been successfully used in long-term epidemiological studies (27, 45) and in deciphering the population structure (2, 20, 28, 45) of Campylobacter on a global scale. MLST has high discriminatory power (23) and standardized nomenclature for STs and clonal complexes (CCs) (3, 26).The aim of our study was to analyze by MLST the overlap and dynamics of C. jejuni types among isolates collected from domestically acquired sporadic human infections from the Helsinki-Uusimaa region from 1996 to 2006 and isolates collected from domestic chicken production from 1999 to 2007.     

Surrogate Antigen Processing Mediated by TAP-dependent Antigenic Peptide Secretion

MHC class I proteins assemble with peptides in the ER. The peptides are predominantly generated from cytoplasmic proteins, probably by the action of the proteasome, a multicatalytic proteinase complex. Peptides are translocated into the ER by the transporters associated with antigen processing (TAP), and bind to the MHC class I molecules before transport to the cell surface. Here, we use a new functional assay to demonstrate that peptides derived from vesicular stomatitis virus nucleoprotein (VSV-N) antigen are actively secreted from cells. This secretion pathway is dependent on the expression of TAP transporters, but is independent of the MHC genotype of the donor cells. Furthermore, the expression and transport of MHC class I molecules is not required. This novel pathway is sensitive to the protein secretion inhibitors brefeldin A (BFA) and a temperature block at 21°C, and is also inhibited by the metabolic poison, azide, and the protein synthesis inhibitor, emetine. These data support the existence of a novel form of peptide secretion that uses the TAP transporters, as opposed to the ER translocon, to gain access to the secretion pathway. Finally, we suggest that this release of peptides in the vicinity of uninfected cells, which we term surrogate antigen processing, could contribute to various immune and secretory phenomena.Protein secretion has traditionally been thought to involve the action of the translocon located in the membrane of the ER of eukaryotic cells. Proteins are recognized cotranslationally when a signal sequence or a signal–anchor sequence emerges from the ribosome (Walter and Johnson, 1994). These sequences are recognized and bound by the signal recognition particle, and the resulting ribosomal complex then interacts with the signal recognition particle receptor on the ER membrane at the translocon (Andrews and Johnson, 1996). This results in the inclusion of proteins within the secretory pathway. This pathway is by far the best described route of protein secretion in eukaryotic cells. Recently, it has been proposed that some proteins are recognized by a component of the translocon, sec 61, exit the ER, and are transported into the cytoplasm where they are degraded (Wiertz et al., 1996).The translocation into the ER of antigenic peptides for presentation by major histocompatibility complex (MHC)¹ class I molecules is largely independent of the translocon. This form of translocation involves the action of two gene products that are members of the ATP binding cassette family. These genes encode transporters associated with antigen processing 1 and 2 (TAP-1 and -2), and have been implicated in the translocation of peptides from the cytoplasm to the lumen of the ER (Deverson et al., 1990; Bahram et al., 1991; Spies and DeMars, 1991; Spies et al., 1992; Gabathuler et al., 1994). After translocation into the ER, antigenic peptides bind to MHC class I molecules composed of a heavy chain (46-kD) and a light chain (12-kD) called β₂m (Nuchtern et al., 1989; Yewdell and Bennink, 1989; van Bleek and Nathenson, 1990; Matsumura et al., 1992), before transport to the cell surface. The assembly and transport of MHC class I molecules appears to be regulated by a series of chaperones that includes calnexin (Degen and Williams, 1991), calreticulin, and tapasin (Sadasivan et al., 1996).High performance liquid chromatography analysis of peptides eluted from acid-treated whole cells or MHC class I molecules has allowed the identification and characterization of the peptides associated with MHC class I molecules (Falk et al., 1990; Rötzschke et al., 1990; van Bleek and Nathenson, 1990). It is proposed that MHC class I molecules determine the final identity of MHC- restricted peptides and have an instructive role, in addition to a selective role, in peptide selection (Wallny et al., 1992). MHC binding to larger peptides followed by protected proteolytic trimming is a possible mechanism that could account for the observed MHC dependency of cellular peptides (Falk et al., 1990). Peptides unable to bind MHC class I because they are in excess or lack the correct MHC binding motif for the MHC haplotype are thus far undetectable by the techniques commonly used in the field, and are presumed to be short lived and degraded (Falk et al., 1990; Rötzschke et al., 1990). Recent results, however, suggest that peptides not able to bind to a MHC class I molecule intracellularly may be found bound to heat shock proteins (HSPs) such as gp96 (grp94; Arnold et al., 1995). These authors show that cellular antigens are represented by peptides associated with gp96 molecules independently of the MHC class I expressed, confirming earlier results (Udono and Srivastava, 1993, 1994). Gp96 extracted from a specific cell is able to induce cross-priming (Udono and Srivastava, 1993, 1994). Finally, two studies have demonstrated that peptides transported into the lumen of the ER, and do not bind to MHC class I molecules, can be transported out of the ER into the cytoplasm again by a process called “efflux” (Momburg et al., 1994; Schumacher et al., 1994), which may involve the action of the TAP molecules or the sec 61 protein associated with the translocon (Wiertz et al., 1996).We have developed a new bioassay to test the hypothesis that peptides translocated into the ER by the action of the TAP molecules become secreted. Using this assay, we present evidence of an alternative secretion pathway that exists in various mammalian cell types. These observations revise the model of peptide catabolism, and may provide an explanation for various immune and secretion phenomena.     

A Ubiquitin E2 Variant Protein Acts in Axon Termination and Synaptogenesis in Caenorhabditis elegans

In the developing nervous system, cohorts of events regulate the precise patterning of axons and formation of synapses between presynaptic neurons and their targets. The conserved PHR proteins play important roles in many aspects of axon and synapse development from C. elegans to mammals. The PHR proteins act as E3 ubiquitin ligases for the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK) to regulate the signal transduction cascade. In C. elegans, loss-of-function of the PHR protein RPM-1 (Regulator of Presynaptic Morphology-1) results in fewer synapses, disorganized presynaptic architecture, and axon overextension. Inactivation of the DLK-1 pathway suppresses these defects. By characterizing additional genetic suppressors of rpm-1, we present here a new member of the DLK-1 pathway, UEV-3, an E2 ubiquitin-conjugating enzyme variant. We show that uev-3 acts cell autonomously in neurons, despite its ubiquitous expression. Our genetic epistasis analysis supports a conclusion that uev-3 acts downstream of the MAPKK mkk-4 and upstream of the MAPKAPK mak-2. UEV-3 can interact with the p38 MAPK PMK-3. We postulate that UEV-3 may provide additional specificity in the DLK-1 pathway by contributing to activation of PMK-3 or limiting the substrates accessible to PMK-3.CHEMICAL synapses are specialized cellular junctions that enable neurons to communicate with their targets. An electrical impulse causes calcium channel opening and consequently stimulates synaptic vesicles in the presynaptic terminals to fuse at the plasma membrane. Neurotransmitter activates receptors on the postsynaptic membrane and triggers signal transduction in the target cell. For this communication to occur efficiently, the organization of the proteins within these juxtaposed pre- and postsynaptic terminals must be tightly regulated (Jin and Garner 2008). Previous studies in Caenorhabditis elegans have identified RPM-1, a member of the conserved PHR (Pam/Highwire/RPM-1) family of proteins, as an important regulator for the synapse (Schaefer et al. 2000; Zhen et al. 2000). Recent functional studies of other PHR proteins have shown that they are also required for a number of steps during nervous system development including axon guidance, growth, and termination (Wan et al. 2000; D''souza; et al. 2005; Bloom et al. 2007; Grill et al. 2007; Lewcock et al. 2007; Li et al. 2008).The signaling cascades regulated by the PHR proteins have been identified using genetic modifier screens (Diantonio et al. 2001; Liao et al. 2004; Nakata et al. 2005; Collins et al. 2006) and biochemical approaches (Grill et al. 2007; Wu et al. 2007). These studies reveal that a major function of PHR proteins is to act as ubiquitin E3 ligases (Jin and Garner 2008). In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) regulates the abundance of its substrate, the dual-leucine-zipper-bearing MAP kinase kinase kinase (DLK MAPKKK), and controls the activity of the MAP kinase cascade composed of three additional kinases, MAPKK MKK-4, p38 MAPK PMK-3, and MAPKAPK MAK-2 (Nakata et al. 2005; Yan et al. 2009). This signaling cascade further regulates the activity of the CCAAT/enhancer binding protein (C/EBP), CEBP-1, via a mechanism involving 3′-UTR-mediated mRNA decay.Signal transduction involving MAP kinases can be fine tuned using multiple mechanisms to ensure optimal signaling outputs (Raman et al. 2007). For example, scaffold proteins for MAP kinases can provide spatial regulation of kinase activation in response to different stimuli (Remy and Michnick 2004; Whitmarsh 2006). Small protein tags such as ubiquitin have also been shown to control the activation of kinases. Specifically, in the IKK pathway ubiquitination via Lys63 chain formation catalyzed by the Ubc13/Uev1a E2 complex and TRAF6 E3 ligase is required for TAK1 kinase activation (Skaug et al. 2009).To further the understanding of the DLK-1 pathway in the development of the nervous system, we characterized a new complementation group of rpm-1(lf) suppressors. These mutations affect the gene uev-3, a ubiquitin E2 conjugating (UBC) enzyme variant (UEV). UEV proteins belong to the UBC family, but lack the catalytic active cysteine necessary for conjugating ubiquitin (Sancho et al. 1998). The best characterized UEV proteins are yeast Mms2 and mammalian Uev1A, both of which act as the obligatory partner for the active E2 Ubc13 and function in DNA repair and IKB pathways, respectively (Deng et al. 2000; Hurley et al. 2006). In addition, UEV proteins, such as Tsg101, can also regulate endosomal trafficking (Babst et al. 2000). We find that similar to other members of the DLK-1 pathway, uev-3 functions cell autonomously in neurons. uev-3 genetically acts downstream of mkk-4 and upstream of mak-2. UEV-3 can bind PMK-3 in heterologous protein interaction assays. We hypothesize that UEV-3 may add specificity to the DLK-1 pathway by binding to PMK-3 for its activation or for selecting specific downstream targets.     

Epitope mapping of Streptococcus mutans SR protein and human IgG cross-reactive determinants, by using recombinant proteins and synthetic peptides.

alpha-Hemolytic oral streptococci are known to possess a family of cell surface cross-reactive proteins termed Ag I/II, having a molecular mass of approximately 180 to 210 kDa. These proteins are implicated in bacterial adherence to various oral tissues, and we showed recently that the SR protein, an I/II Ag-related protein, from Streptococcus mutans OMZ 175 serogroup f possesses Ag mimicry with human IgG. In this study, regions of the SR protein encoding the cross-reactive epitope(s) were analyzed by expressing selected restriction fragments from the cloned sr gene. The three SR-derived polypeptides reacted in ELISA with anti-SR rabbit IgG, whereas only the two polypeptides located along the carboxyl-terminal two thirds of the SR protein reacted with anti-human IgG rabbit IgG. In order to locate more precisely the human IgG-cross-reactive region, we synthesized six peptides, on the basis of the recently determined complete nucleotide sequence of the sr gene. Among these peptides, peptide 2, corresponding to the alanine-rich repeating amino-terminal region, peptide 3, located in the three tandem proline-rich regions, and peptide 6, located near the cell wall-spanning region, were the most interesting in term of antigenicity and immunogenicity. Anti-peptide 2, 3, and 6 rabbit IgG reacted with free SR and with cell wall-associated SR. Peptide 1, located near the amino terminus, was poorly immunogenic. Peptides 4 and 5, located in the putative human IgG-cross-reactive region, were immunogenic; however, anti-peptide 4 rabbit IgG reacted only weakly with SR or human IgG, whereas anti-peptide 5 rabbit IgG reacted strongly with SR and human IgG, and peptide 5 was recognized by anti-SR and anti-human IgG rabbit IgG. These results confirm the cell surface accessibility of this epitope and its potential participation in eliciting, in rabbits, anti-SR IgG cross-reactive with human IgG.     

ChiloKey,an interactive identification tool for the geophilomorph centipedes of Europe (Chilopoda,Geophilomorpha)

ChiloKey is a matrix-based, interactive key to all 179 species of Geophilomorpha (Chilopoda) recorded from Europe, including species of uncertain identity and those whose morphology is known partially only. The key is intended to assist in identification of subadult and adult specimens, by means of microscopy and simple dissection techniques whenever necessary. The key is freely available through the web at: http://www.biologia.unipd.it/chilokey/ and at http://www.interactive-keys.eu/chilokey/.     

RCSB Protein Data bank: Tools for visualizing and understanding biological macromolecules in 3D

Now in its 52nd year of continuous operations, the Protein Data Bank (PDB) is the premiere open‐access global archive housing three‐dimensional (3D) biomolecular structure data. It is jointly managed by the Worldwide Protein Data Bank (wwPDB) partnership. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) is funded by the National Science Foundation, National Institutes of Health, and US Department of Energy and serves as the US data center for the wwPDB. RCSB PDB is also responsible for the security of PDB data in its role as wwPDB‐designated Archive Keeper. Every year, RCSB PDB serves tens of thousands of depositors of 3D macromolecular structure data (coming from macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro‐electron diffraction). The RCSB PDB research‐focused web portal (RCSB.org) makes PDB data available at no charge and without usage restrictions to many millions of PDB data consumers around the world. The RCSB PDB training, outreach, and education web portal (PDB101.RCSB.org) serves nearly 700 K educators, students, and members of the public worldwide. This invited Tools Issue contribution describes how RCSB PDB (i) is organized; (ii) works with wwPDB partners to process new depositions; (iii) serves as the wwPDB‐designated Archive Keeper; (iv) enables exploration and 3D visualization of PDB data via RCSB.org; and (v) supports training, outreach, and education via PDB101.RCSB.org. New tools and features at RCSB.org are presented using examples drawn from high‐resolution structural studies of proteins relevant to treatment of human cancers by targeting immune checkpoints.     

NNAlign_MA; MHC Peptidome Deconvolution for Accurate MHC Binding Motif Characterization and Improved T-cell Epitope Predictions

1. Download : Download high-res image (106KB)
2. Download : Download full-size image

Highlights

• •NNAlign_MA enables full deconvolution of single MHC specificities from MS assays.
• •NNAlign_MA expands MHC allelic coverage, improving identification of T-cell epitopes.
• •NNAlign_MA was benchmarked on MHC classes I and II, outperforming current methods.
• •NNAlign_MA offers a universal solution to analyze and exploit MHC peptidomics data.