High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases.

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue-and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C.

Hepatic inactivation of murine results in marked reduction in plasma cholesterol.

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018).

Identification of novel γ-globin inducers among all potential erythroid druggable targets.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals.

Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape.

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system.