A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

A High-Throughput System for Transient and Stable Protein Production in Mammalian Cells.

Recombinant protein expression and purification is an essential component of biomedical research and drug discovery. Advances in automation and laboratory robotics have enabled the development of highly parallel and rapid processes for cell culture and protein expression, purification, and analysis. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells have emerged as the standard host cell workhorses for producing recombinant secreted mammalian proteins by using both transient and stable production strategies. In this chapter we describe a fully automated custom platform, Protein Expression and Purification Platform (PEPP), used for transient protein production from HEK cells and stable protein production from CHO cells. Central to PEPP operation is a suite of custom robotic and instrumentation platforms designed and built at GNF, custom cell culture ware, and custom scheduling software referred to as Runtime. The PEPP platform enables cost-effective, facile, consistent production of proteins at quantities and quality useful for early stage drug discovery tasks such as screening, bioassays, protein engineering, and analytics.

Mammalian cell culture density determination using a laser through-beam sensor.

High-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation and proteomics, as well as for direct use as potential biotherapeutic agents for medical applications. Here we describe a device that we refer to as the flask density reader (FDR) consisting of a through-beam laser and sensor, and a customized culture flask-receiving nest. The FDR has been integrated onto GNF System™'s automated protein expression platform to enable rapid, noninvasive, fully automated spectrophotometric determination of cell densities in suspension mammalian cell cultures. The FDR reduces the risk of culture contamination from frequent flask sampling and greatly reduces the time and effort needed to count cells using off-line methods.

Identification of a candidate therapeutic antibody for treatment of canine B-cell lymphoma.

B-cell lymphoma is one of the most frequently observed non-cutaneous neoplasms in dogs. For both human and canine BCL, the standard of care treatment typically involves a combination chemotherapy, e.g. "CHOP" therapy. Treatment for human lymphoma greatly benefited from the addition of anti-CD20 targeted biological therapeutics to these chemotherapy protocols; this type of therapeutic has not been available to the veterinary oncologist. Here, we describe the generation and characterization of a rituximab-like anti-CD20 antibody intended as a candidate treatment for canine B-cell lymphoma. A panel of anti-canine CD20 monoclonal antibodies was generated using a mouse hybridoma approach. Mouse monoclonal antibody 1E4 was selected for construction of a canine chimeric molecule based on its rank ordering in a flow cytometry-based affinity assay. 1E4 binds to approximately the same location in the extracellular domain of CD20 as rituximab, and 1E4-based chimeric antibodies co-stain canine B cells in flow cytometric analysis of canine leukocytes using an anti-canine CD21 antibody. We show that two of the four reported canine IgG subclasses (cIgGB and cIgGC) can bind to canine CD16a, a receptor involved in antibody-dependent cellular cytotoxicity (ADCC). Chimeric monoclonal antibodies were assembled using canine heavy chain constant regions that incorporated the appropriate effector function along with the mouse monoclonal 1E4 anti-canine CD20 variable regions, and expressed in CHO cells. We observed that 1E4-cIgGB and 1E4-cIgGC significantly deplete B-cell levels in healthy beagle dogs. The in vivo half-life of 1E4-cIgGB in a healthy dog was ∼14 days. The antibody 1E4-cIgGB has been selected for further testing and development as an agent for the treatment of canine B-cell lymphoma.

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies.

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.

Two novel WD40 domain-containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway.

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway.

Novel Ist1-Did2 complex functions at a late step in multivesicular body sorting.

In Saccharomyces cerevisiae, integral plasma membrane proteins destined for degradation and certain vacuolar membrane proteins are sorted into the lumen of the vacuole via the multivesicular body (MVB) sorting pathway, which depends on the sequential action of three endosomal sorting complexes required for transport. Here, we report the characterization of a new positive modulator of MVB sorting, Ist1. We show that endosomal recruitment of Ist1 depends on ESCRT-III. Deletion of IST1 alone does not cause cargo-sorting defects. However, synthetic genetic analysis of double mutants of IST1 and positive modulators of MVB sorting showed that ist1Delta is synthetic with vta1Delta and vps60Delta, indicating that Ist1 is also a positive component of the MVB-sorting pathway. Moreover, this approach revealed that Ist1-Did2 and Vta1-Vps60 compose two functional units. Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and, like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provide evidence that the ist1Delta mutation exhibits a synthetic interaction with mutations in VPS2 (DID4) that compromise the Vps2-Vps4 interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60 complexes independently modulate late steps in the MVB-sorting pathway.

An internal ribosome entry site promotes translation of a novel SIV Pr55(Gag) isoform.

In complex retroviruses including simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), the major structural proteins are encoded by the gag gene and translated as a precursor polyprotein, Pr55(Gag). An internal ribosome entry site (IRES) within the coding region of HIV-1 and HIV type 2 (HIV-2) gag RNA mediates expression of N-terminally truncated isoforms of the precursor polyprotein. In this study, we identify an N-terminally truncated SIV Pr55(Gag) isoform expressed from the SIV gag gene SIV p43. We demonstrate that translation of p43 occurs independently of Pr55(Gag) translation and initiates at an in-frame AUG within the gag transcript. We test several mechanisms that could mediate translation of p43 and report that translation of SIV p43 is driven by an IRES located entirely within the coding region of gag mRNA. Additionally, we present data that suggest SIV p43 affects viral replication in cell culture.

Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus.

The lentiviral Gag polyprotein (Pr55(Gag)) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55(Gag) is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55(Gag) and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55(Gag), several CA-containing cleavage intermediates of Pr55(Gag), and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55(Gag) can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55(Gag) and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55(Gag) and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.

Conserved serines in simian immunodeficiency virus capsid are required for virus budding.

The simian immunodeficiency virus (SIV) capsid protein (CA), a constituent of the Pr55Gag polyprotein, is phosphorylated in virions but not in virus-producing cells (Rue, S.M., Roos, J.W., Tarwater, P.M., Clements, J.E., Barber, S.A., 2005. Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus. J. Virol. 79 (4), 2484-2492.). Using phosphoamino acid analysis of CA, we show that serine is the primary phosphate acceptor. A series of substitution mutants of serines in the CA domain of Pr55Gag were constructed in the infectious viral clone SIVmac239. These virus mutants were examined for defects in virus replication and virion infectivity, release, and morphology, as well as alterations in phosphorylation of CA-containing proteins. Although the virus mutants exhibited a number of replication defects, none of these defects could be directly attributed to aberrant CA phosphorylation. A novel defect was a block in early budding, which was common among several virus mutants with substitutions in the CA N terminus. Together, these results indicate that certain residues in the CA N terminus are crucial for early budding events.

Hydrogen bonding at a conserved threonine in lentivirus capsid is required for virus replication.

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55(Gag) polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T(47)(CA), that is requisite for viral replication. Replacement of T(47)(CA) in the infectious viral clone SIVmac239 with amino acids with different hydrogen-bonding capabilities and analysis of the effects of these substitutions at key steps in the viral life cycle demonstrate that hydrogen bonding at this position is important for virus infectivity and virion release. In the HIV-based homology model of the mature SIV CA N terminus presented in this study, T(47)(CA) forms several hydrogen bonds with a proximal aspartate, D(50)(CA). This model, coupled with strong phenotypic similarities between viral substitution mutants of each of these two residues in all of the virological assays described herein, indicates that hydrogen bonding between T(47)(CA) and D(50)(CA) is likely required for viral replication. As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.