Anti-ATF5 (internal region) polyclonal antibody

Trypanosoma cruzi, the causative agent of Chagas' disease, infects heart and muscle cells leading to cardiac arrest, followed by death. The genetic architectures in the early T. cruzi infection process of human cells are unknown. To understand the genetic architectures of the early invasion process of T. cruzi, we conducted gene transcription microarray analysis, followed by gene network construction of the host cell response in primary human coronary artery smooth muscle (HCASM) cells infected with T. cruzi or exposed to T. cruzi gp83, a ligand used by the trypanosome to bind host cells. Using seven RT-PC12 verified up-regulated genes (FOSB, ATF5, INPP1, CCND2, THBS1, LAMC1, and APLP2) as the seed for network construction, we built an interaction network of the early T. cruzi infection process containing 165 genes, connected by 598 biological interactions. This interactome network is centered on the BCL6 gene as a hub. Silencing the expression of two seed genes (THBS1 and LAMC1) by RNAi reduced T. cruzi infection. Overall, our results elucidate the significant and complex process involved in T. cruzi infection of HCASM cells at the transcriptome level. This is the first elucidation into the interactome network in human cells caused by T. cruzi and its gp83 ligand.

Amorphous aggregation is a major problem for protein biopharmaceuticals, and aggregate formation in a drug formulation can have serious health implications for the patient. In many cases, an immunogenic response is generated from the administration of a drug product containing aggregated protein. This becomes especially significant when the patient requires long-term or repeated administration of the drug, because the likelihood of a severe immune response increases. While the prevention of protein aggregation is critically important for the future of protein pharmaceuticals, the mechanism of amorphous aggregation is still poorly understood. The lack of understanding regarding nonfibrillar aggregation is largely due to the fact that assembly is difficult to study. In particular the role that various structural features (i.e., a-helix, P-structure, disulfide bonds) play in the aggregation process varies with the amino acid sequence and is dependent upon tertiary structure and solution conditions. Well-structured proteins do not readily aggregate in solution, whereas partially unfolded proteins tend to aggregate rapidly and often become insoluble. Here, we present a unique and simple system for studying amorphous protein aggregation. We have previously reported the isolation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5), a protein notable for its potential as a pharmaceutical target for treatment of glioblastoma multiforme. This domain consists of a single a-helix and possesses a single cysteine residue. It is only partially structured and displays marginal stability in solution under physiological conditions. We have modulated solution conditions that affect backbone solubility and the oxidation state of the thiol to successfully investigate the role that a-helical structure and disulfide bond formation play in protein stability. Our data indicate that covalent cross-linking helps to retain ATF5's helicity, which inhibits the formation of large aggregates. These studies have led to the identification of stabilizing conditions for ATF5, which will enable further study of the protein as a pharmaceutical target. Moreover, this work has general implications for analyzing stability of helical proteins in vitro as well as the specific atomic-level interactions in ATF5 that contribute to instability and self -association.