Exploring proteoforms of the IgG2 monoclonal antibody panitumumab using microchip capillary electrophoresis-mass spectrometry.

The IgG2 type monoclonal antibody panitumumab is an anti-epidermal growth factor receptor (EGFR) drug used for the treatment of EGFR-expressing, chemotherapy resistant, metastatic colorectal carcinoma. In this study, panitumumab drug product was first analysed using size exclusion chromatography coupled to mass spectrometry for rapid identity testing. The experimental data led to the identification of two panitumumab isoforms with several prominent forms remaining unidentified, despite apparently low sample complexity.

Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells.

Threonine Phosphorylation of IκBζ Mediates Inhibition of Selective Proinflammatory Target Genes.

Transcription factors of the NF-κB family play a crucial role for immune responses by activating the expression of chemokines, cytokines, and antimicrobial peptides involved in pathogen clearance. IκBζ, an atypical nuclear IκB protein and selective coactivator of particular NF-κB target genes, has recently been identified as an essential regulator for skin immunity. This study discovered that IκBζ is strongly induced in keratinocytes that sense the fungal glucan zymosan A.

Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination.

Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk.

Exploring host-pathogen interactions through genome wide protein microarray analysis.

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins.

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S.

Protectome analysis: a new selective bioinformatics tool for bacterial vaccine candidate discovery.

New generation vaccines are in demand to include only the key antigens sufficient to confer protective immunity among the plethora of pathogen molecules. In the last decade, large-scale genomics-based technologies have emerged. Among them, the Reverse Vaccinology approach was successfully applied to the development of an innovative vaccine against Neisseria meningitidis serogroup B, now available on the market with the commercial name BEXSERO® (Novartis Vaccines).

Mining the bacterial unknown proteome: identification and characterization of a novel family of highly conserved protective antigens in Staphylococcus aureus.

In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S.

Staphylococcus aureus FhuD2 is involved in the early phase of staphylococcal dissemination and generates protective immunity in mice.

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface.

Streptococcus pyogenes SpyCEP: a chemokine-inactivating protease with unique structural and biochemical features.

SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids.

Surfome analysis as a fast track to vaccine discovery: identification of a novel protective antigen for Group B Streptococcus hypervirulent strain COH1.

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family.

Proteomics characterization of outer membrane vesicles from the extraintestinal pathogenic Escherichia coli DeltatolR IHE3034 mutant.

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant.

Proteomic analysis of the airway surface liquid: modulation by proinflammatory cytokines.

The airway surface is covered by a fluid, the airway surface liquid, interposed between the mucous layer and the epithelium. The airway surface liquid contains proteins, secreted by different cell types, that may have pro-/anti-inflammatory or bactericidal functions or have a role in the mucociliary clearance. We have used a proteomics approach to identify the proteins secreted by an isolated in vitro model of human airway epithelium, at resting and under proinflammatory conditions, as a strategy to define the factors involved in epithelial barrier function.

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome.

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature.

Gelsolin secretion in interleukin-4-treated bronchial epithelia and in asthmatic airways.

Rationale: The airway surface liquid, the thin layer of liquid covering the airways, is essential for mucociliary clearance and as a barrier against microbial and other noxious agents. Proteins secreted into the airway surface liquid by epithelial and nonepithelial cells may be important in innate immunity and to improve the fluidity of mucous secretions.

Objectives: We aimed to identify proteins specifically secreted into the airway surface liquid by human bronchial epithelial cells, in resting conditions and after treatment with interleukin 4 (IL-4), a cytokine released in asthma.

Discovering high mobility group A molecular partners in tumour cells.

DNA-based activities rely on an extremely coordinated sequence of events performed by several chromatin-associated proteins which act in concert. High Mobility Group A (HMGA) proteins are non-histone architectural nuclear factors that participate in the regulation of specific genes but they are also believed to have a more general role in chromatin dynamics. The peculiarity of these proteins is their flexibility, both in terms of DNA-binding and in protein-protein interactions.

Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis.

The aim of this study was to analyze the type of immune response (Th1, Th2) and protein composition of bronchoalveolar lavage (BAL) of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Flow cytometry analysis of intracellular cytokines revealed different patterns: in IPF and SSc Th2 profiles were predominant, whereas in sarcoidosis Th1 prevailed. The proteomic analysis of BAL fluid (BALF) showed that there were quantitative differences between the three diseases.

Immunoblotting techniques.

Immunoblotting techniques use antibodies (or other specific ligands in related techniques) to identify target proteins among a number of unrelated protein species. They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions. Proteins are typically separated by electrophoresis and transferred onto membranes (usually nitrocellulose).

Biochemical characterization of the THIN-B metallo-beta-lactamase of Janthinobacterium lividum.

The THIN-B metallo-beta-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa.

Induction of Mycobacterium avium proteins upon infection of human macrophages.

Induction of Mycobacterium avium proteins labelled with [35S]methionine and mRNAs upon infection of the human macrophage cell line THP-1 was investigated by two-dimensional gel electrophoresis-mass spectrometry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. M. avium overexpressed proteins within the macrophages that are involved in fatty acids metabolism (FadE2, FixA), cell wall synthesis (KasA), and protein synthesis (EF-tu).

Proteomic approach to the identification of voltage-dependent anion channel protein isoforms in guinea pig brain synaptosomes.

Voltage-dependent anion channel (VDAC) proteins are small, abundant, pore-forming proteins belonging to the eukaryotic mitochondrial porins. At least three different VDAC genes have been identified in vertebrates. VDAC proteins are known to play an essential role in cellular metabolism and in the early stages of apoptosis.

Translationally controlled tumor protein (TCTP) in the human prostate and prostate cancer cells: expression, distribution, and calcium binding activity.

Background: The translationally controlled tumor protein (TCTP) is an abundantly expressed protein found in a wide range of organisms from both the animal and plant kingdom. Initially described as a growth-related protein, knowledge of the biological actions of TCTP has been recently extended to include calcium binding, regulation of apoptosis, and microtubules stabilization. This report describes expression, distribution, and characterization of TCTP in human prostatic tissues and cell lines.

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells.