Analytical and Functional Similarity of the Biosimilar Candidate ABP 654 to Ustekinumab Reference Product.

Background And Objective: ABP 654 is a proposed biosimilar to ustekinumab reference product (RP), a human immunoglobulin isotype class G subclass 1 kappa monoclonal antibody that acts as an antagonist of interleukin (IL)-23 and IL-12. Ustekinumab RP is indicated for the treatment of some forms of plaque psoriasis, active psoriatic arthritis, Crohn's disease, and ulcerative colitis. ABP 654 and ustekinumab RP utilize different expression systems, and the purpose of this study was to assess analytical similarity between ABP 654 and ustekinumab RP sourced from the United States (US) and the European Union (EU).

Analytical and Functional Similarity Assessment of ABP 710, a Biosimilar to Infliximab Reference Product.

Purpose: ABP 710 has been developed as a biosimilar to infliximab reference product (RP). The objective of this study was to assess analytical similarity (structural and functional) between ABP 710 and infliximab RP licensed by the United States Food and Drug Administration (infliximab [US]) and the European Union (infliximab [EU]), using sensitive, state-of-the-art analytical methods capable of detecting minor differences in product quality attributes.

Methods: Comprehensive analytical characterization utilizing orthogonal techniques was performed with 14 to 28 unique lots of ABP 710 or infliximab RP, depending on the assay.

Nonsense-mediated decay regulates key components of homologous recombination.

Cells frequently experience DNA damage that requires repair by homologous recombination (HR). Proteins involved in HR are carefully coordinated to ensure proper and efficient repair without interfering with normal cellular processes. In Saccharomyces cerevisiae, Rad55 functions in the early steps of HR and is regulated in response to DNA damage through phosphorylation by the Mec1 and Rad53 kinases of the DNA damage response.

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.

dFMRP and Caprin, translational regulators of synaptic plasticity, control the cell cycle at the Drosophila mid-blastula transition.

The molecular mechanisms driving the conserved metazoan developmental shift referred to as the mid-blastula transition (MBT) remain mysterious. Typically, cleavage divisions give way to longer asynchronous cell cycles with the acquisition of a gap phase. In Drosophila, rapid synchronous nuclear divisions must pause at the MBT to allow the formation of a cellular blastoderm through a special form of cytokinesis termed cellularization.

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression [corrected].

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions.

A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1.

CDC25B mediates rapamycin-induced oncogenic responses in cancer cells.

Because the mammalian target of rapamycin (mTOR) pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anticancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an as yet unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin.

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum.

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins.

Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC.

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts.

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins.

Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry.

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.

Fragile X mental retardation protein controls trailer hitch expression and cleavage furrow formation in Drosophila embryos.

During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. Here, we demonstrate that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition.

Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway.

Protein phosphorylation has become a focus of many proteomic studies due to the central role that it plays in biology. We combine peptide-based gel-free isoelectric focusing and immobilized metal affinity chromatography to enhance the detection of phosphorylation events within complex protein samples using LC-MS. This method is then used to carry out a quantitative phosphoproteomic analysis of the tumor necrosis factor (TNF) pathway using HeLa cells metabolically labeled with 15N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway.

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol.

Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast.

The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs. The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms. Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway.

Strategies for shotgun identification of post-translational modifications by mass spectrometry.

The global identification of post-translationally modified proteins is a difficult challenge that is currently being addressed by many researchers in the field of mass spectrometry (MS)-based proteomics. The ability to identify thousands of proteins by shotgun-based strategies has made the mere idea of a global analysis of a particular protein modification seem reasonable. There has been much progress in the development of methods that make use of shotgun-based protein identification in the analysis of a wide variety of protein modifications, some of which will be discussed here.

Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA.

The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2-/- embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2-/- cells.

Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit.

Sur2 is a metazoan Mediator subunit that interacts with the adenovirus E1A protein and functions in a mitogen-activated protein kinase pathway required for vulva development in Caenorhabditis elegans. We generated sur2-/- embryonic stem cells to analyze its function as a mammalian Mediator component. Our results show that Sur2 forms a subcomplex of the Mediator with two other subunits, TRAP/Med100 and 95.