Liquid-liquid phase separation promotes the formation of membraneless condensates that mediate diverse cellular functions, including autophagy of misfolded proteins. However, how phase separation participates in autophagy of dysfunctional mitochondria (mitophagy) remains obscure. We previously discovered that nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) translocates from the nucleus to mitochondria to mediate celastrol-induced mitophagy through interaction with p62/SQSTM1. Here, we show that the ubiquitinated mitochondrial Nur77 forms membraneless condensates capable of sequestrating damaged mitochondria by interacting with the UBA domain of p62/SQSTM1. However, tethering clustered mitochondria to the autophagy machinery requires an additional interaction mediated by the N-terminal intrinsically disordered region (IDR) of Nur77 and the N-terminal PB1 domain of p62/SQSTM1, which confers Nur77-p62/SQSTM1 condensates with the magnitude and liquidity. Our results demonstrate how composite multivalent interaction between Nur77 and p62/SQSTM1 coordinates to sequester damaged mitochondria and to connect targeted cargo mitochondria for autophagy, providing mechanistic insight into mitophagy.
Recombinant tissue-type plasminogen activator (r-tPA) was approved by U.S. Food and Drug Administration as a thrombolytic drug. However, a high dose of r-tPA (up to 100 mg/person) is typically used in clinical applications. Such high dosage leads to severe side effects including haemorrhage and neurotoxicity, which can be fatal. To improve the proteolytic properties of tPA to enhance thrombolytic therapy, we designed a series of mutants in tPA serine protease domain (tPA-SPD) based on the crystal structure of tPA-SPD:plasminogen activators inhibitor-1 (PAI-1) complex that we determined recently. We found that the A146Y substitution in tPA-SPD(A146Y) enhanced resistance to PAI-1 inactivation by 30-fold compared with original tPA-SPD. Interestingly, the tPA-SPD(A146Y) variant showed fivefold higher activation for plasminogen compared with tPA-SPD. The variant also demonstrated thrombolytic activity stronger than tPA-SPD in a clot lysis assay. In vivo, we showed tPA-SPD(A146Y) possessed higher thrombolytic efficacy in a pulmonary embolism model compared with original tPA-SPD. Furthermore, a mouse tail bleeding assay showed that tPA-SPD(A146Y) did not increase bleeding risk compared with clinical drug r-tPA. Together, our findings reveal novel functions of A146Y variant, which not only increases the catalytic efficiency of the enzyme, but also enhances resistance to PAI-1 inhibition, and demonstrating that tPA-SPD (A146Y) variant is a much improved agent for thrombolytic therapy.
Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.
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