Advantages of extended bottom-up proteomics using sap9 for analysis of monoclonal antibodies.

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

"In vitro" reconstitution of the fragmentation of vacuoles

Résumé La fragmentation des membranes est un processus commun à beaucoup d'organelles dans une cellule. Les mitochondries, le noyau, le réticulum endoplasmique, les phagosomes, les peroxisomes, l'appareil de Golgi et les lysosomes (vacuoles chez la levure) se fragmentent en plusieurs copies en réponse à des sitmulis environnementaux, tels que des stresses, ou dans une situtation normale durant le cycle cellulaire, afin d' être transférer dans les cellules filles. La fragmentation des membranes est également observée pendant le processus d'endocytose, lors de la formation de vésicules endocytiques, mais également dans tout le traffic intracellulaire, lors de la genèse d'une vésicule de transport. Le processus de fragmentation est donc généralement important. La découverte en 1991 d'une dynamin-like GTPase comme protéine impliquée dans la fragmentation de la membrane plasmique durant l'endocytose a ouvert ce domaine de recherche. Dès lors des dynamines ont été découvertes sur la pluspart des organelles, ce qui suggère un processus de fragmentation des membranes commun à l'ensemble de la cellule. Cependant, l'ensemble des protéines impliquées ainsi que le mécanisme de la fragmentation reste encore à élucider. Mon projet de thèse était d'établir un test in vitro de fragmentation des vacuoles utile à la compréhension du mécanisme de ce processus. Le choix de ce système est judicieux pour plusieurs raisons; premièrement les vacuoles fragmentent naturellement durant le cycle cellulaire, deuxièment leur taille permet de visualiser facilement leur morphologie par simple microscopie optique, finalement elles peuvent être isolées en quantité intéressante avec un haut degré de pureté. In vivo, les vacuoles peuvent être facilement fragmentées par un stress osmotique. Un tel test permet d'identifier des protéines impliquées dans le mécanisme comme dans le criblage que j'ai effectué sur l'ensemble de la collection de délétions des gènes non-essentiels chez la levure. Cependant un test in vitro est ensuite indispensable pour jouer avec les protéines découvertes afin d'en élucider le mécanisme. Avec mon test in vitro, j'ai confirmé l'implication des protéines SNAREs dans la fragmentation et j'ai permis de comprendre la régulation de la quantité de vacuoles et de leur taille par le complexe TORC1 dans une situation de stress. 7 Résumé large public Les cellules de chaque organisme sont composées de différents compartiments appelés organelles. Chacun possède une fonction bien définie afin de permettre la vie et la croissance de la cellule. Ils sont entourés de membrane, qui joue le role de barrière spécifiquement perméable, afin de garder l'intégrité de chacun. Dans des conditions de croissance normale, les cellules prolifèrent. Durant la division cellulaire amenant à la formation d'une nouvelle cellule, chaque organelle doit se diviser afin de fournir l'ensemble des organelles à la cellule fille. La division de chaque organelle nécessite la fragmentation de la membrane les entourant. Des protéines dynamine-like GTPase ont été découvertes sur presque l'ensemble des organelles d'une cellule. Elles sont impliquées dans les processus de fragmentation des membranes. Dès lors l'idée d'un mécanisme commun est apparu. Cependant cette réaction, par sa complexité, ne peut pas impliquer une protéine unique. La découverte d'autres facteurs et la compréhension du mécanisme reste à faire. La première étape peut se faire par étude in vivo, c'est-à-dire avec des cellules entières, la deuxième étape, quant à elle, nécessite d'isoler les protéines impliquées et de jouer avec les différents paramètres, ce qui signifie donc un travail in vitro, séparé des cellules. Mon travail a constisté à établir un procédé expérimental in vitro pour étudier la fragmentation des membranes. Je travaille avec des vacuoles de levures pour étudier les réactions membranaires. Les vacuoles sont les plus grandes organelles présentes dans les levures. Elles sont impliquées principalement dans la digestion. Comme toute organelle, elles se fragmentent durant la division cellulaire. Le procédé expérimental comporte une première étape, l'isolation des vacuoles et, deuxièmement, l'incubation de celles-ci avec des composés essentiels à la réaction. En parallèle, j'ai mis en évidence, par un travail in vivo, de nouvelles protéines impliquées dans le processus de fragmentation des membranes. Ceci a été fait en réalisant un criblage par microscopie d'une collection de mutants. Parmi ces mutants, j'ai cherché ceux qui présentaient un défaut dans la fragmentation des vacuoles. Ces deux procédés expérimentaux, in vitro et in vivo, m'ont permis de découvrir de nouvelles protéines impliquées dans cette réaction, ainsi que de mettre en évidence un mécanisme utlilisé par la cellule pour réguler la fragmentation des vacuoles. 8 Summary Fragmentation of membranes is common for many organelles in a cell. Mitochondria, nucleus, endoplasmic reticulum, phagosomes, peroxisomes, Golgi and lysosomes (vacuoles in yeast) fragment into multiple copies in response to environmental stimuli, such as stresses, or in a normal situation during the cell cycle in order to be transferred into the daughter cell. Fragmentation of membrane occurs during endocytosis, at the latest step in endocytic vesicle formation, and also in intracellular trafficking, when traffic vesicles bud. This field of research was opened in 1991 when a dynamin-like GTPase was found to be involved in fragmentation of the plasma membrane during endocytosis. Since dynamin-like GTPases have been found on most organelles, similarities in their mechanisms of fragmentation might exist. However, many proteins involved in the mechanism of fragmentation remain unknown. My thesis project was to establish an in vitro assay for membrane fragmentation in order to create a tool to study the mechanism of this process. I chose vacuoles as a model organelle for several reasons: first of all, vacuoles fragment under physiological conditions during cell cycle, secondly their size makes their morphology easily visible under the light microscope, and finally vacuoles can be isolated in good amounts with relatively high degrees of purity. In vivo, vacuole fragmentation can be induced with an osmotic shock. Such a simple assay facilitates the identification of new proteins involved in the process. I used this tool to screen of the entire knockout collection of non-essential genes in Saccharomyces cerevisiae for mutants defective in vacuole fragmentation. The in vitro system will be useful to characterize the mutants and to study the mechanism of fragmentation in detail. I used my in vitro assay to confirm the involvement of vacuolar SNARE proteins in fragmentation of the organelle and to uncover that number and size of vacuoles in the cell is regulated by the TORC1 complex via selective stimulation of fragmentation activity.

HER2 shedding and serum HER2 extracellular domain: Biology and clinical utility in breast cancer.

The transmembrane protein HER2 is over-expressed in approximately 15% of invasive breast cancers as a result of HER2 gene amplification. HER2 proteolytic cleavage (HER2 shedding) generates soluble truncated HER2 molecules that include only the extracellular domain and the concentration of which can be measured in the serum fraction of blood. HER2 shedding also generates a constitutively active truncated intracellular receptor of 95kDa (p95(HER2)). Another soluble truncated HER2 protein (Herstatin), which can also be found in serum, is the product of an alternatively spliced HER2 transcript. Recent preclinical findings may provide crucial insights into the biological and clinical relevance of increased sHER2 concentrations for the outcome of HER2-positive breast cancer and sensitivity to trastuzumab and lapatinib treatment. We present here the most recent findings about the role and biology of sHER2 based on data obtained using a standardized test, which has been cleared by FDA in 2000, for measuring sHER2. This test includes quality control assessments and has been already widely used to evaluate the clinical utility of sHER2 as a biomarker in breast cancer. We will describe in detail data concerning the assessment of sHER2 as a surrogate maker to optimize the evaluation of the HER2 status of a primary tumor and as a prognosis and predictive marker of response to therapies, both in early and metastatic breast cancer.

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface.

The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

Current drug discovery strategies against arenavirus infections.

Arenaviruses are a large group of emerging viruses including several causative agents of severe hemorrhagic fevers with high mortality in man. Considering the number of people affected and the currently limited therapeutic options, novel efficacious therapeutics against arenaviruses are urgently needed. Over the past decade, significant advances in knowledge about the basic virology of arenaviruses have been accompanied by the development of novel therapeutics targeting different steps of the arenaviral life cycle. High-throughput, small-molecule screens identified potent and broadly active inhibitors of arenavirus entry that were instrumental for the dissection of unique features of arenavirus fusion. Novel inhibitors of arenavirus replication have been successfully tested in animal models and hold promise for application in humans. Late in the arenavirus life cycle, the proteolytic processing of the arenavirus envelope glycoprotein precursor and cellular factors critically involved virion assembly and budding provide further promising 'druggable' targets for novel therapeutics to combat human arenavirus infection.

Obstructive fibrostenotic Crohn's disease.

Crohn's disease is often complicated by gastrointestinal strictures. Postoperative recurrence at the anastomotic site is common and repeated surgical interventions may be necessary. Medical treatment may relieve active inflammation (see chapter on active luminal disease) but fibrous strictures will not respond to this. Mechanical treatment methods consist of endoscopic balloon dilation, stricturoplasty or surgical resection. Fibrostenotic Crohn's disease does not respond to medical therapy and requires endoscopic or surgical treatment.

Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence.

Fungal infections represent a serious threat, particularly in immunocompromised patients. Interleukin-1beta (IL-1beta) is a key pro-inflammatory factor in innate antifungal immunity. The mechanism by which the mammalian immune system regulates IL-1beta production after fungal recognition is unclear. Two signals are generally required for IL-1beta production: an NF-kappaB-dependent signal that induces the synthesis of pro-IL-1beta (p35), and a second signal that triggers proteolytic pro-IL-1beta processing to produce bioactive IL-1beta (p17) via Caspase-1-containing multiprotein complexes called inflammasomes. Here we demonstrate that the tyrosine kinase Syk, operating downstream of several immunoreceptor tyrosine-based activation motif (ITAM)-coupled fungal pattern recognition receptors, controls both pro-IL-1beta synthesis and inflammasome activation after cell stimulation with Candida albicans. Whereas Syk signalling for pro-IL-1beta synthesis selectively uses the Card9 pathway, inflammasome activation by the fungus involves reactive oxygen species production and potassium efflux. Genetic deletion or pharmalogical inhibition of Syk selectively abrogated inflammasome activation by C. albicans but not by inflammasome activators such as Salmonella typhimurium or the bacterial toxin nigericin. Nlrp3 (also known as NALP3) was identified as the critical NOD-like receptor family member that transduces the fungal recognition signal to the inflammasome adaptor Asc (Pycard) for Caspase-1 (Casp1) activation and pro-IL-1beta processing. Consistent with an essential role for Nlrp3 inflammasomes in antifungal immunity, we show that Nlrp3-deficient mice are hypersusceptible to Candida albicans infection. Thus, our results demonstrate the molecular basis for IL-1beta production after fungal infection and identify a crucial function for the Nlrp3 inflammasome in mammalian host defence in vivo.

Drug safety in the treatment of Crohn's disease.

The management of Crohn's disease usually consists of a succession of short-term acute phase treatments followed by long-term maintenance therapy. The disease affects young patients and for this reason the long-term safety of the drugs needs to be especially taken into consideration. The safety, dose, duration for optimal efficacy and the most frequent adverse events will be described in this article.

Purification of recombinant proteins by chemical removal of the affinity tag.

The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.

Final Report for the Iowa Livestock Industry Waste Characterization and Methane Recovery Information Dissemination Project, 2003

The Iowa livestock industry generates large quantities of manure and other organic residues; composed of feces, urine, bedding material, waste feed, dilution water, and mortalities. Often viewed as a waste material, little has been done to characterize and determine the usefulness of this resource. The Iowa Department of Natural Resources initiated the process to assess in detail the manure resource and the potential utilization of this resource through anaerobic digestion coupled with energy recovery. Many of the pieces required to assess the manure resource already exist, albeit in disparate forms and locations. This study began by interpreting and integrating existing Federal, State, ISU studies, and other sources of livestock numbers, housing, and management information. With these data, models were analyzed to determine energy production and economic feasibility of energy recovery using anaerobic digestion facilities on livestock faxms. Having these data individual facilities and clusters that appear economically feasible can be identified specifically through the use of a GIs system for further investigation. Also livestock facilities and clusters of facilities with high methane recovery potential can be the focus of targeted educational programs through Cooperative Extension network and other outreach networks, providing a more intensive counterpoint to broadly based educational efforts.

Dietary supplementation with omega-3 in cystic fibrosis patients

Accumulation of neutr ophils releasing proteolytic enzymes and free radicals induceprogressive lung tissue destruction in CF. Among several hfflammatory mediatorsimplicated in this process, leukotriene B 4 (LTB4) product of arachidonic (AA;20:4n 6) omega 6 polytmsaturated fatty acid (PUFA) plays an important role.Various anti inflammatory strategies including dietary supplementation of omega 3PUFA, known to favor the synthesis of less active leukotriene B 5 (LTBs), have beeninvestigated. To further explore this nutritional approach, biological effects of anomega 3 PUFA oral supplementation (n 3 OS) were measured in 17 CF patients haa prospective, randomized, double blind, crossover study. CF patients (mean age:18 + 9 years, FEVI: 66 + 29 %) received a dietary supplementation enriched, or not,ha omega 3 PUFA during a 2 x 6 months period. A modification in neutrophilmembrane PUFA composition was observed under n 3 OS with an increase in EPA(20:5n 3) PUFA (from 0.66 ± 0.56 to 1.60 ± 0.61 ~tmol %, P< 0.01). The LTB jLTB 5ratio was decreased (from 72 + 27 to 24 + 7, P< 0.C~31), in CF patients taking n 3OS. However, n 3 OS supplementation did neither affect the internalization of bothIL 8 receptors following IL 8 exposure, nor IL 8 induced neutrophil chemotaxis.Our results show that n 3 PUFA are absorbed and incorporated in neutrophilmembrmae. The consecutive decrease ha LTBjLTB 5 ratio suggests that, ha theseconditions, neutr ophils may produce less toxic mediators from the AA pathway. Thepotential clinical benefit for CF patients still needs to be assessed with furtherstudies of longer duration and including more patients.

Treatment of postoperative Crohn's disease.

At 1 year after a first resection, up to 80% of patients show an endoscopic recurrence, 10-20% have clinical relapse, and 5% have surgical recurrence. Smoking is one of the most important risk factors for postoperative recurrence. Preoperative disease activity and the severity of endoscopic lesions in the neoterminal ileum within the first postoperative year are predictors of symptomatic recurrence. Mesalamine is generally the first-line treatment used in the postoperative setting but still provokes considerable controversy as to its efficacy, in spite of the results of a meta-analysis. Immunosuppressive treatment (azathioprine, 6-MP) is based on scant evidence but is currently used as a second-line treatment in postsurgical patients at high risk for recurrence, with symptoms or with early endoscopic lesions in the neoterminal ileum. Nitroimidazole antibiotics (metronidazole, ornidazole) are also effective in the control of active Crohn's disease in the postoperative setting. Given their known toxicity, they may be used as a third-line treatment as initial short-term prevention therapy rather than for long-term use. Conventional corticosteroids, budesonide or probiotics have no proven role in postoperative prophylaxis. Infliximab has not as yet been studied for use in the prevention of relapse after surgery.

Antigenicity and immunogenicity of a novel Plasmodium vivax circumsporozoite derived synthetic vaccine construct.

BACKGROUND: The circumsporozoite (CS) protein is a major malaria sporozoite surface antigen currently being considered as vaccine candidate. Plasmodium vivax CS (PvCS) protein comprises a dimorphic central repeat fragment flanked by conserved regions that contain functional domains involved in parasite invasion of host cells. The protein amino (N-terminal) flank has a cleavage region (region I), essential for proteolytic processing prior to parasite invasion of liver cells. METHODS: We have developed a 131-mer long synthetic polypeptide (LSP) named PvNR1R2 that includes the N-terminal flank and the two natural repeat variant regions known as VK210 and VK247. We studied the natural immune response to this region in human sera from different malaria-endemic areas and its immunogenicity in mice. RESULTS: PvNR1R2 was more frequently recognized by sera from Papua New Guinea (PNG) (83%) than by samples from Colombia (24%) when tested by ELISA. The polypeptide formulated in Montanide ISA51 adjuvant elicited strong antibody responses in both C3H and CB6F1 mice strains. Antibodies from immunized mice as well as affinity-purified human IgG reacted with native protein by IFA test. Moreover, mouse immune sera induced strong (90%) in vitro inhibition of sporozoite invasion (ISI) of hepatoma cell lines. CONCLUSIONS: These results encourage further studies in non-human primates to confirm the elicitation of sporozoite invasion blocking antibodies, to assess cell mediated immune responses and the protective efficacy of this polypeptide.

Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

Oxidized LDL modulates apoptosis of regulatory T cells in patients with ESRD.

Increased levels of oxidized low-density lipoproteins (oxLDL) contribute to the increased risk for atherosclerosis, which persists even after adjusting for traditional risk factors, among patients with ESRD. Regulatory T cells (CD4+/CD25+ Tregs), which down-regulate T cell responses to foreign and self-antigens, are protective in murine atherogenesis, but whether similar immunoregulation occurs in humans with ESRD is unknown. Because cellular defense systems against oxLDL involve proteolytic degradation, the authors investigated the role of oxLDL on proteasome activity of CD4+/CD25+ Tregs in patients with ESRD. CD4+/CD25+ Tregs isolated from uremic patients' peripheral blood, especially that of chronically hemodialyzed patients, failed to suppress cell proliferation, exhibited cell-cycle arrest, and entered apoptosis by altering proteasome activity. Treating CD4+/CD25+ Tregs with oxLDL or uremic serum ex vivo decreased the number and suppressive capacity of CD4+/CD25+ Tregs. In vitro, oxLDL promoted the accumulation of p27Kip1, the cyclin-dependent kinase inhibitor responsible for G1 cell cycle arrest, and increased apoptosis in a time- and concentration-dependent manner. In summary, proteasome inhibition by oxLDL leads to cell cycle arrest and apoptosis, dramatically affecting the number and suppressive capacity of CD4+/CD25+ Tregs in chronically hemodialyzed patients. This response may contribute to the immune dysfunction, microinflammation, and atherogenesis observed in patients with ESRD.