The progesterone receptor (PR) occurs in two major forms, the full-length PRB and the amino-truncated PRA, which lacks 164 amino-terminal residues. PRB functions as a strong transcriptional activator of progesterone-responsive genes, whereas PRA is inactive in several cell types where it may even act as a trans-dominant repressor of PRB and other steroid receptors, like the glucocorticoid receptor or, reportedly, the estrogen receptor. We initially observed that a PR deleted of its entire amino domain (PR538-C) is incapable of trans-repressing PRB or glucocorticoid receptor, suggesting that a negative modulation domain must be contained in the region between position 165 and 538. After testing progressive deletion mutants and chimeras, we demonstrate that this negative modulating domain is confined within 120 residues in the amino-terminal region and that it contains a subdomain of 40 residues that is crucial for intermolecular transrepression. Duplication, deletion, and transplantation of the negative modulation domain show that the negative modulation domain has only a limited functional autonomy. In our hands, transrepression of estrogen receptor could not be substantiated, and, under our conditions, at least an equimolar concentration of PRA expression plasmid is required for transrepression. Our deletion studies reveal domains that correlate with strong homology patches between the amino-terminal domains of mammalian and avian PR.

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Transepithelial water flow across the renal proximal tubule is mediated predominantly by aquaporin-1 (AQP1). Along this nephron segment, luminal delivery and transepithelial reabsorption are directly coupled, a phenomenon called glomerulotubular balance. We hypothesized that the surface expression of AQP1 is regulated by fluid shear stress, contributing to this effect. Consistent with this finding, we found that the abundance of AQP1 in brush border apical and basolateral membranes was augmented .2-fold by increasing luminal perfusion rates in isolated, microperfused proximal tubules for 15 minutes. Mouse kidneys with diminished endocytosis caused by a conditional deletion of megalin or the chloride channel ClC-5 had constitutively enhanced AQP1 abundance in the proximal tubule brush border membrane. In AQP1-transfected, cultured proximal tubule cells, fluid shear stress or the addition of cyclic nucleotides enhanced AQP1 surface expression and concomitantly diminished its ubiquitination. These effects were also associated with an elevated osmotic water permeability. In sum, we have shown that luminal surface expression of AQP1 in the proximal tubule brush border membrane is regulated in response to flow. Cellular trafficking, endocytosis, an intact endosomal compartment, and controlled protein stability are the likely prerequisites for AQP1 activation by enhanced tubular fluid shear stress, serving to maintain glomerulotubular balance.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that γδ T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites. Here, we follow up on the molecular mechanisms of parasite growth inhibition by human pore-forming proteins. We confirm that Plasmodium falciparum infection efficiently depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, by revealing the molecular events we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.