Pathways of Proteins

We begin with a brief summary of the degradation pathways of proteins, followed by a discussion on the composition of liquid and lyophilized protein formulations and on various excipients in some detail. An important feature of this chapter is a comprehensive table (Appendix), which details the formulations of approved protein drugs through the year 2005. The table has been compiled with the help of several sources (1,10,11). 

Proteins can degrade via the physical processes of interfacial adsorption and aggregation. Proteins are surface-active molecules, i.e., they tend to adsorb at liquid solid, liquid air, and liquid-liquid interfaces. It is well established that proteins fold into their unique three-dimensional structures, which consist of a hydrophobic core 

Proteins are subject to a variety of chemical modification/degradation reactions, viz. deamidation, isomerization, hydrolysis, disulfide scrambling, beta-elimination, and oxidation. The principal hydrolytic mechanisms of degradation include peptide bond 

Beta-elimination reactions have been observed in a number of proteins. This reaction occurs primarily at alkaline pH conditions. Abstraction of the hydrogen atom from the alpha-carbon of a cysteine, serine, threonine, phenylalanine, or lysine residue leads to racemization or loss of part of the side chain and the formation of dehydroalanine (26). 

The stability of proteins toward covalent degradation pathways can often depend on the protein s folded state. In each pathway, solvent accessibility and varying degrees of structural freedom of the peptide backbone and/or side chains around the labile residue are required for reactions to take place. Accordingly, stabilization of the protein s folded state (i.e., its compact structure) that minimizes solvent accessibility can lower the reaction rate of some covalent protein modifications, extending the shelf life of the protein product. Therefore, the selection of formulation excipients depends on their direct and indirect influence on the rates of covalent protein degradation. 

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Matthews, C.R. Pathways of protein folding. Anna. Rev. Biochem. 62 653-683, 1993. 

Weissman, J.S. All the roads lead to Rome The multiple pathways of protein folding. Chem. Biol. 2 255-260, 1995. 

The above describes the major pathway of proteins destined for the mitochondrial matrix. However, certain proteins insert into the outer mitochoiidrial membrane facilitated by the TOM complex. Others stop in the intermembrane space, and some insert into the inner membrane. Yet others proceed into the matrix and then return to the inner membrane or intermembrane space. A number of proteins contain two signaling sequences—one to enter the mitochondrial matrix and the other to mediate subsequent relocation (eg, into the inner membrane). Certain mitochondrial proteins do not contain presequences (eg, cytochrome Cy which locates in the inter membrane space), and others contain internal presequences. Overall, proteins employ a variety of mechanisms and routes to attain their final destinations in mitochondria. 

The pathways of protein import into mitochondria, nuclei, peroxisomes, and the endoplasmic reticulum are described. 

T. J. Ahern and M. J. Manning, Stability of Protein Pharmaceuticals, Part A, Chemical and Physical Pathways of Protein Degradation, Plenum Press, New York, 1992. 

Fersht, A. R., Matouschek, A., and Serrano, L. (1992). The folding of an enzyme. 1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771-782. 

One suggestive piece of support that leads to the idea that certain different RNA informed pathways of protein synthesis from amino acids arose in different vesicles is the correlation between the different first and, to some degree, the second... 

Saraste, J. and Kuismanen, E. Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex. Semin. Cell Biol. 3 343-355,1992. 

Ito, K. (1996). The major pathways of protein translocation across membranes. Genes Cells 1, 337-346. 

Settles, A., and Martienssen, R. (1998). Old and new pathways of protein export in chloroplasts and bacteria. Trends Cell Biol. 8, 494-501. 

Hilser, V.J. and E. Freire. 1996. Structure-based calculation of the equilibrium folding pathway of proteins. Correlation with hydrogen exchange protection factors. 

Varshavsky, A. The N-end rule pathway of protein degradation. Genes CeUs 1997, 2, 13-28. 

Lee DH, Goldberg AL (1996) Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae. J Biol Chem 271 27280-27284... 

THE LIPID PATHWAY OF PROTEIN GLYCOSYLATION AND ITS INHIBITORS THE BIOLOGICAL SIGNIFICANCE OF PROTEIN-BOUND CARBOHYDRATES... 

In the first Section, the dolichol pathway of protein glycosylation is introduced, and the reader is made familiar with the various reactions in the formation of the lipid and carbohydrate moieties of lipid-linked saccharides. Three different classes of compound are known so far (a) isoprenoid alcohol esters of monosaccharide monophosphates, such as D-mannosyl and D-glucosyl (dolichol phosphate), (b) such isoprenoid alcohol esters of saccharide diphosphates as dolichol diphosphate linked to 2-acetamido-2-deoxy-D-glucose and to oligosaccharides, and (c) retinol (D-mannosyl phosphate). The dolichol-linked sugars occur in all eukaryotes. 

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