Citrullinemia type I (CTLN-I) results from the absence or deficiency of argininosuccinate synthetase (AS), a 46 kDa enzyme that acts in the cytosol of hepatocytes to convert aspartic acid and citrulline into argininosuccinic acid. AS is an essential component of the urea cycle, and its absence or deficiency results in the harmful accumulation of ammonia in blood and cerebrospinal fluid. No disease-modifying treatment of CTLN-I exists. Here we report that the cell-permeant miniature protein (CPMP) ZF5.3 (ZF) can deliver AS to the cytosol of cells in culture and the livers of healthy mice. The fusion protein ZF-AS is catalytically active in vitro, stabilized in plasma, and traffics successfully to the cytosol of cultured Saos-2 and SK-HEP-1 cells, achieving cytosolic concentrations greater than 100 nM. This value is 3-10-fold higher than the concentration of endogenous AS (11 ± 1 to 44 ± 5 nM). When injected into healthy C57BL/6 mice, ZF-AS reaches the mouse liver to establish concentrations almost 200 nM above baseline. These studies demonstrate that ZF5.3 can deliver a complex enzyme to the cytosol at therapeutically relevant concentrations and support its application as an improved delivery vehicle for therapeutic proteins that function in the cytosol, including enzyme replacement therapies.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.