Protein inactivation pathways

The protein-C pathway is one of the most important anticoagulant mechanisms. It is activated by thrombin. Thrombin binds to a cofactor in the membrane of endothelial cells, thrombomodulin (TM). TM bound thrombin no longer activates clotting factors or platelets but becomes an effective protein C (PC) activator. Activated PC (APC) forms a complex with Protein S, which inactivates FVIIIa and FVa. Hereby generation of Flla by the prothrombinase complex is inhibited (Fig. 9). Thus, the PC-pathway controls thrombin generation in a negative feedback manner. 

A type I transmembrane protein called endothelial cell protein C receptor (EPCR), which is expressed at high levels exclusively on a subset of endothelial cells, has also been identified. EPCR has a role in the protein C pathway (30). EPCR binds to both protein C and activated protein C (APC) with equal affinity. Activation of protein C presumably requires interaction of the protein C-EPCR complex with the thrombin-thrombomodulin complex. APC that is formed as a result of this interaction is reversibly bound to EPCR until it dissociates to react subsequently with protein S. The APC-protein S complex inactivates activated factor V (Va). 

Fig. 7. Our current understanding of the protein C pathway. V, and VIII, represent inactivated factors V and VIII, respectively. For an explanation of other abbreviations see text.

The Ras pathway is shown here. Ras is a G-protein that couples signaling from growth factors. The activated receptor is a GNRP that increases the exchange of GDP for GTP and activates the G-protein. Ras GAP inactivates the G-protein. The downstream signal for activated Ras is eventually the mitogen-activated protein kinase pathway (MAPK). 

Even with the knowledge of the reactive moieties that are suspected to trigger M BI, there are numerous potential pathways for the chemistry to lead to protein inactivation [174,196]. Differentiating these mechanisms can facilitate the generation of alternate and safer chemical scaffolds. The UV-Vis spectrophotometer has been a key instrument in activity and functional characterization for CYPs for the past 40 years, as indicated by the derivation of its name, pigment 45 0 being the signature UV band present when reduced in the presence of CO [5,197]. This technique has been... 

Fig. 1.57. Model of the regulation of translation by insulin. Insulin ( and other growth factors) activates the Akt kinase pathway (see ch. 10), whose final result is the phosphorylation of 4E-BPl, a regulatory protein of translation initiation. The 4E-BP1 protein inactivates the initation factor eIF-4E by complex formation. eIE-4E is required, together with the proteins eIE-4A and p220, for the binding of the 40S subunit of the ribosome to the cap structure of the mRNA. If the 4E-BP1 protein becomes phosphorylated as a result of insulin-mediated activation of the PI3 kinase/Akt kinase cascade, then eIE-4E is liberated from the inactive eIP-4E 4E-BPl complex and protein biosynthesis can begin.

Although aggregation is the predominant means by which proteins become inactivated during refolding, several other inactivation pathways have also been observed. Proteins can be inactivated by thiol-disulfide exchange or alteration of the primary structure by chemical modification of amino acid side chains. In addition, refolded proteins may be inactivate due to the absence of prosthetic groups and metals or because of improper association of the subunits in multimeric proteins (79). 

Tumor suppressor genes like p53, Rb, and E2P appeared during evolution, probably to protect the integrity of the organism from uncontrolled cell proliferation within multicellular specialized tissues. This concept is supported by the observation that the respective proteins or pathways are mutated or inactivated in most human cancers. Cell cycle entry (GO-Gl- to S-phase transition) is controlled by two major pathways (1) the Rb (Rb, cy-clin Dl, and pl6 ° ) cell cycle pathway and (2) the p53/ p2iwafi Q2-S checkpoint arrest pathway. Although both pathways act largely independently, they are interconnected (Burke et al. 2005 Hsieh et al. 2002). 

Ironically, while water is critical in maintaining molecular shape, the aqueous state is not one in which proteins are long resistant to denaturation. A variety of environmental changes such as temperature, pH, salts and solvents can cause protein inactivation in the aqueous state, and the mechanisms of irreversible protein inactivation often follow conunon pathways. These include cystein destruction, thiol-catalyzed disulfide interchange, oxidation of cystein residues, deamidation of asparagine and glutamine residues and hydrolysis of peptide bonds at aspartic acid residues. 

Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASKl/Jun N-terminal protein kinase pathway normally activated at G(2)/M. 

In about 30-40% of patients with suspected inherited thrombophilia the PC-pathway is disturbed by a mutation ofFV (FV-Leiden). TheFV-Leiden mutation affects one of the APC cleavage sites within the FV molecule. As a consequence, mutated FVa becomes resistant to rapid APC inactivation (APC resistance). About 4-7% of the middle European population cany this polymorphism of FV. Inborn deficiencies of Protein-S or Protein-C are much less frequent (< < 1% and 0.2-0.4%, respectively). 

Because of the multiple degradation pathways that may take place at elevated temperature, protein stability monitoring data may not conform to the Arrhenius relationship, and the maximum temperature selected for accelerated stability studies must be carefully selected. Gu et al. [32] described the different mechanisms of inactivation of interleukin-1 (3 (IL-1 (3) in solution above and below 39°C. In this example, the multiple mechanisms precluded the prediction of formulation shelf life from accelerated temperature data. In contrast, by working at 40° C and lower, Perlman and Nguyen [33] were able to successfully extrapolate data from stability studies of tissue plasminogen activator down to 5°C. 

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