Protein Inhibitors:

Function and Role

 

Table of Contents

Introduction

Overview

Introduction

As seen in the previous section, protein synthesis inhibitors are an extremely diverse and ubiquitous class of biochemical molecules. Their function ranges from bacterial translation inhibition to blocking synthesis of specific amino acid residues necessary for the production of a protein. The role of inhibitors in organisms is a huge one and has served as a key to answering many questions in the fields of biochemistry and medicine. The material in this document is meant to focus on two typical mechanisms of protein synthesis inhibition, both antibiotics, and an unusual case of of inhibition which shows promise of fighting a debilitating disease.

Overview

1.) Antibiotics

2.) Posttranslational Inhibition


Antibiotics

In general, antibiotics are biochemically or fungally produced substances that inhibit the growth of other organisms. Most antibiotics, like many pharmaceuticals, block translation in protein synthesis. These substances are effective because they take advantage of the tremendous complexity involved in the synthesis of proteins. The following is a table of some of these synthesis inhibitors, the systems they act on, and their mode of action. Click on any inhibitor name to view its structure.

Name of Inhibitor

Sytems Inhibited

Mode of Action

Tetracycline (1)

prokaryotic

Inhibits aminoacyl-tRNA binding

Penicillin

prokaryotic

Inhibits transpeptidation enzymes involved in the cross-linking of polysacchride chains of the bacterial cell wall peptidoglycan

Chloramphenicol (1)

prokaryotic

Binds to 50S subunit, blocks peptidyl transferase activity

Erythromycin (1)

prokaryotic

Binds to 50S subunit, blocks peptidyl transferase activity

Rifampin

prokaryotic

Blocks RNA synthesis by binding to and inhibiting the DNA-dependent RNA polymerase

Cycloheximide (1)

eukaryotic

Inhibits translocation of peptidyl-tRNA

Bacitran

prokaryotic

Inhibits cell wall synthesis by interferring with action of the lipid carrier that transports wall precursors accross plasma membrane.

Fusidic acid (1)

both

Inhibits EF-G:GDP dissociation from ribosome

Thiostrepton (1)

prokaryotic

Inhibits ribosome-dependent EF-Tu and EF-G GTPase

Puromycin (1)

both

Aminoacyl-tRNA analog, which acts as a peptidyl acceptor, aborting further peptide elongation

Sulfonamides

prokaryotic

Inhibit folic acid synthesis by competition with p-aminobenzoic acid

Streptomycin (1)

prokaryotic

codon misreading, insertion of improper amino acid Prevents formation of initiation complex

It is important to note that protein synthesis in eukaryotes and prokaryotes proceeds through the same steps of initiation, elongation, and termination. These general similarities in mechanism allow certain antibiotics to act on both systems effectively, often making the substance an excellent tool in research.

 

Puromycin

Puromycin, whose chemical structure was first noted in 1950 by Yarmolinsky and de la Haba, is an antibiotic critical to many studies because of its effect on both prokaryotic and eukaryotic cells. (2) .It is a structural analog of the aminoacyl-adenylyl grouping characteristic of the 3' -end of aminoacyl tRNAs. One of the most striking differences between the two molecules is the adenine moiety carries two methyl groups on its amino nitrogen, and the tyrosyl residue, which forms the amino acid residue, is also methylated in its phenolic oxygen group (2). Another important difference is that instead of an ester linkage attaching the amino acyl grouping, puromycin uses an amide grouping.

 

Puromycin inhibits protein synthesis at translation by prematurely terminating a peptide chain. In simple terms, the part of puromycin that resembles an aminoacyl end of tRNA can bind to the A site of a ribosome, (forming a peptide bond), but the end product will not participate in translocation to the P site. Specifically, puromycin serves as an acceptor of the peptidyl chain from peptidyl-tRNA in the P site, in a reaction in which peptidyl transferase catalyzes the attachment of the peptidyl chain to the free NH3+ group of puromycin (1). Notice that this analog is not linked by the usual ester bond (to the 3'-OH terminus in all tRNAs), but rather via an amide bond to the 3'-NH.

(1)

This lack of participation in translocation results in dissociation from the ribosome and early peptide termination. This early termination means the release of truncated, nonfunctional polypeptides. For two animations, one demonstrating normal polypeptide formation, and one demonstrating polypeptide formation involving puromycin, click on the icon below.

The reasons for this late stage inhibition lie in experimental results which examined the mechanism of puromycin. In the protein synthesizing system which uses amino acyl-tRNA and ribosomes, compounds containing the amino acids originally attached to tRNA appear in an alcohol-soluble fraction of the incubation mixture. It is suggested that these alcohol-soluble materials are peptides or fragments of incomplete peptides released for the ribosome by puromycin.

Because of its effects on both prokaryotes and eukaryotes, puromycin is an extremely important component in a variety of studies. Some of these studies include the detailing of proteolytic pathways that recognize and destroy abnormal or truncated proteins. By adding puromycin, researchers can demonstrate its rapid degradation by a ubiquitin-dependent pathway. This can lead to theories regarding why other proteins, which are vulnerable to proteolysis, are not degraded (3). Another study involved the demonstration of protein-translocating aqueous pores in the ER membrane. Using puromycin to release growing peptides from a ribosome, researchers may explore the possibilities of polypeptide chains transferring across the ER membrane either in direct contact with the lipid bilayer or through a pore in a protein translocator (3).

Streptomycin

In modern medicine, most of the effective antibiotics are compounds that specifically inhibit bacterial protein synthesis. By taking advantage of the structural differences between prokaryotic and eukaryotic ribosomes, doctors can administer high doses of drugs with no worry of toxicity to human patients. One of these drugs is streptomycin, discovered in 1944 by Selman Waksman and since then included in the family of antibiotics known as aminoglycosides. Streptomycin is produced by the actinomycete Streptomyces griseus. Waksman received the Nobel Prize in 1952, and his success led to a worldwide search for other antibiotic-producing siol microorganisms(5).

Streptomyces griseus. Colonies of the actinomycete that produces streptomycin.(5)

Depending on its concentration, streptomycin can affect bacterial cells in two ways. At low concentrations, it induces mRNA misreading, so that improper amino acids are incorporated into the polypeptide. Codons with pyrimidines in the first and second positions are particularly susceptible to streptomycin-induced misreading (1). Because these mistakes in reading are not frameshift errors, completely abnormal proteins are not made at low streptomycin levels. This results in a severely depressed growth rate, not death, in susceptible cells. At high concentrations, 70s nonproductive ribosome: mRNA complexes accumulate, preventing formation of active initiation complexes with new mRNA (1). This prevention of proper chain initiation causes cell death.

Certain streptomycin resistant mutants (strR) have ribosomes with an altered protein S12 compared with streptomycin sensitive bacteria (strS). Intriguingly, a change in base C912 of 16S or rRNA also confers streptomycin resistance (1). (Some mutant bacteria are not only resistant to streptomycin, but are dependent on it; they require it for growth.) In partial diploid bacteria that are heterozygous for streptomycin resistance, (strR/strS), streptomycin sensitivity is dominant. This puzzling observation is explained by finding that, in the presence of streptomycin, strS ribosomes remain bound to initiation sites thereby excluding strR ribosomes from these sites. Moreover, the mRNAs in these blocked complexes are degraded after a few minutes, which allows the strS ribosomes to bind to newly synthesized mRNA as well (1).

Other Antibiotic Uses and Resistance

Eventhough antibiotics are extremely important in the treatment of diseases, it should also be noted that many antibiotics make excellent research tools. For example, they aid in the cultivation of viruses by preventing bacterial contamination.. When eggs are inoculated with a virus sample, antibiotics are often included in the inoculum to maintain sterility. Antibiotics often aid researchers as instruments which can dissect metabolic processes by inhibiting or blocking specific steps and observing the consequences (5). Another example may be the administration of antibiotics to a specific cell. If a scientist cared to study the dependence of bacterial flagella synthesis on RNA transcription, the flagella could be removed by a high speed mixer, followed by the addition of actinomycin D to the mixture. The bacterial culture could then be observed for flagella regeneration in the absence of RNA synthesis.

Because of the massive amounts of antibiotics that are being produced and used in diseases, there is an increasing number of drug resistance around the world. This drug resistance is obviously a serious problem which is related to a general misuse of antibiotics. Antibiotics are all too often prescribed without culturing and identifying the pathogen or without determing bacterial sensitivity to the drug. Sometimes toxic, broad-spectrum drugs are given in place of narro-spectrum drugs as a substitute for culture and sensitivity testing. To worsen an already bad situation, many patients do not complete the course of medication, allowing the possibility of drug-resistant mutations to survive.


Postranslational Regulation

Once a polypeptide chain is assembled, it still requires two major "finishing steps" before it becomes functional. These steps are chemical modification and folding. These two steps are quite interrelated, for the final folding pattern of a protein depends largely on the chemically modified portions of the macromolecule. Chemical modification involves three steps: 1) modification of amino acid residues into other types, 2) addition of organic units (such as sugars or lipids) to specific amino acids, 3) enzymatic cleavage of one or more amino acids from a region of the polypeptide chain.(4). The following material will focus on the importance chemical modification, specifically the modification of amino acids into other types, is in normal tissue function.

Scleroderma

The abundance of collagen in the extracellular structures of humans and other mammals makes disorders of collagen deposition and synthesis an important factor in disease.(4) Atherosclerosis, a disease involving stiffening of the arteries, is related to an over-deposition of collagen while fibrosis, or hardening of the tissues, is related to excessive collagen synthesis. Some diseases which involve fibrosis are pulmonary fibrosis, which can severely impair the ability of lungs to expand, and scleroderma (Progressive Systemic Sclerosis). Scleroderma is classified as a disease of the vascular and immune systems, and a severe connective tissue disorder. It results in fibrosis of the skin and multiple organs (6). Individuals afflicted with scleroderma have fibroblasts which produce an excess amount of collagen, this excess of one protein can cause the disease to range from a minor skin lesion to a debilitating and sometimes fatal disease (6).

One of the many regions of the body often affected by scleroderma is the hands. In the pictures above, notice the severe decrease in soft tissue near the bones of the fingers. (6)

One key to controlling scleroderma is controlling the rate of collagen that is produced, and a currently researched method of doing this is the use of 3,4-dihydroxybenzoic acid. Before examining the mechanism behind this substance, it is necessary to understand process by which collagen is synthesized.

Collagen (7,8), which has evolved for strength, has a unique structure consisting of a left-handed triple helix with three amino acid residues per turn. The usual amino acid sequence is gly-x-pro or gly-x-hyp, where x is any amino acid and hyp is hydroxyproline. Hydroxyproline is required for normal formation of the collagen triple helix, and below is a chart which demonstrates its importance. Denaturation of collagen molecules with normal content of hydroxyproline and of abnormal collagen containing no hydroxyproline. Without hydrogen bonds between hydroxyproline residues, the helix is unstable and loses most of its helical contents at temperatures above 20 C. Such collagens are formed by experiment in animals (or man) in the absence of ascorbic acid (vitamin C). Normal collagen is stable and resists denaturation until a temperature of 40 C is reached (7).

(7)

Hydroxyproline is the product of proline residues which undergo hydroxylation in the rough ER. The diagram below illustrates all the required components for this reaction.

(7)

Note that the enzyme prolyl hydroxylase is required for proper hydroxylation, and 4-hydroxyproline is the predominant for of hydroxyproline. Any nonhydroxylated procollagens are degraded within the cell and kept from forming in the triple helix. 3,4-Dihydroxybenzoic acid has been known to act as a potent competitive inhibitor of purified prolyl 4-hydroxylase with respect to one or several of the cofactors or cosubstrates of the enzyme (9).

In one particular study, different modifications of 3,4-dihydroxybenzoic acid were tested in human skin fibroblast cultures for their efficacy to inhibit the synthesis of 4-hydroxyproline. The results indicated that the ethyl ester of 3,4-dihydroxybenzoic acid was an efficient inhibitor of prolyl hydroxylation in fibroblast cultures, with Ki of approximately .4mM. Ethyl 3,4-dihydroxybenzoate had little, if any effect on the hydroxylation of lysyl residues, and it did not affect the total protein synthesis or DNA replication in these cells (9).

To test the hypothesis that ethyl 3,4-dihydroxybenzoate might serve as a potential antifibrotic agent, it's efficacy in inhibiting prolyl hydroxylation in scleroderma fibroblasts was als tested. The results indicated that the synthesis of 4-hydroxyproline in scleroderma cell cultures was similarly reduced by ethyl 3,4-dihydroxybenzoate. Thus, structural analogs of the cofactors or cosubstrates of prolyl 4-hydroxylase, such as ethyl 3,4-dihydroxybenzoate tested here or its further modifications, may serve as inhibitors of posttranslational hydroxylation of prolyl residues also in vivo. These compounds could potentially provide a novel means of reducing collagen deposition in tissues in fibrotic diseases, such as scleroderma (9).

Other methods of treatment, such as Penicillamine, are being used and researched. Penicillamine is a known metal binding, or chelating agent that also interferes with the formation of cross-links between tropocollagen molecules . Certain lysine and hydroxylysine residues are oxidated into reactive aldehydes which spontaneously form specific cross-links between two chains of procollagen. These covalent cross-links are obviously very important to the stability of tropocollagen, and any severe disruption will result in damage to the protein. For an illustration of this process, please view the Collagen Biosynthesis (7) page.

Conclusion

The importance of protein synthesis inhibitors in functioning organisms cannot be stressed enough. The aim of this document was to shed light on the variety of ways that synthesis inhibitors may affect the lives of humans, and to build an appreciation for the enormous diversity and complexity in cells.


References

1.) Garret, R.; Grisham, C. Biochemistry. Austin: Saunders College Publishing; 1995

2.) Ingram, V. Biosynthesis of macromolecules. Menlo Park, California: W.A. Benjamin Inc; 1972

3.) Alberts, B. Molecular biology of the cell. New York: Garland Publishing; 1994

4.) Wolfe, S.L. Molecular and Cellular biology. Belmont, California: Wadsworth Publishing Company; 1993

5.) Prescott, L.M.; Harley, J.P.; Klein, D.A. Microbiology. Boston: Wm. C. Publishers; 1996

6.) United Scleroderma Foundation http://www.scleroderma.com

7.) Darnell, J.; Lodish, H.; Baltimore, D. Molecular cell biology. New York: W.H. Freeman and Company; 1990

8.) Lehninger, A.; Neilson, D.; Cox, M. Principles of biochemistry. New York: Worth Publishers; 1993

9.) Majamaa,K.; Sasaki, T.; Uitto, J. Inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblasts cultures by ethyl 3,4-dihydroxybenzoate. Department of Medicine, UCLA School of Medicine, Torrance


 


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