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Results for “zymogens”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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7 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Zymogen and activated protein C have similar structural architecture

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule Förster resonance energy transfer and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca 2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid EGF domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing EGF domains, such as factors VII, IX, and X.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Massively parallel, computationally guided design of a proenzyme

Proteins have shown promise as therapeutics and diagnostics, but their effectiveness is limited by our inability to spatially target their activity. To overcome this limitation, we developed a computationally guided method to design inactive proenzymes or zymogens, which are activated through cleavage by a protease. Since proteases are differentially expressed in various tissues and disease states, including cancer, these proenzymes could be targeted to the desired microenvironment. We tested our method on the therapeutically relevant protein carboxypeptidase G2 (CPG2). We designed Pro-CPG2s that are inhibited by 80 to 98% and are partially to fully reactivatable following protease treatment. The developed methodology, with further refinements, could pave the way for routinely designing protease-activated protein-based therapeutics and diagnostics that act in a spatially controlled manner. Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1988

Development of immunoassays for human urokinase

Radioimmune assays (RIA) and enzyme linked immune assays for measurement of pro-urokinase and the two active forms of the enzyme were developed. Polyclonal and monoclonal antibodies, with desired specificities against preselected synthetic regions of urokinase (UK), were obtained by immunization with the respective synthetic peptides and used to develop RIA for zymogen and the two activated forms of UK.

Atassi, M. Zouhair↗
NASA NTRS1994

Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

STS-54 Shuttle Project↗
NASA NTRS2007

Antibodies Against Three Forms of Urokinase

Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

Morrison, Dennis R.↗