The Effect of Acetylation and Deacetylation in Post-translational Regulation in Bacteria: Difference between revisions

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==Acetylation in Salmonella enterica==
==Acetylation in Salmonella enterica==
[[Zbc0300513090002.jpeg⎮thumb⎮⎮1800px⎮⎮right⎮Acs proteins in S. enterica are acetylated by Pat, rendering them inactive. The sirtuin cobB deacetylates Acs with the use of NAD+. Once, deacetylated, acetate can be converted to the intermediate acetyl-AMP and then acetyl coenzyme A.]]
[[Zbc0300513090002.jpeg⎮thumb⎮⎮1800px⎮⎮right⎮Acs proteins in S. enterica are acetylated by Pat, rendering them inactive. The sirtuin cobB deacetylates Acs with the use of NAD+. Once, deacetylated, acetate can be converted to the intermediate acetyl-AMP and then acetyl coenzyme A.]]
In the bacteria Salmonella enterica, post-translational modification of protein function has been determined to have a metabolic role by interacting with acetyl-coenzyme A (Ac-CoA) synthetase (Acs), the enzyme responsible for the conversion of acetate to the acetyl-adenosine monophosphate (Ac-AMP) intermediate and then to Ac-CoA (6).  Acetyl-CoA is fundamental to cell metabolism, most importantly in the TCA cycle.  Acetyl CoA is received mostly from the breakdown of pyruvate, but also from other catabolic pathways such as fat, amino acid, and benzoate breakdown.  Acetyl CoA is taken up into the TCA cycle by incorporation via the combination of the acetyl group with oxaloacetyate.  The cycle can then proceed to produce 3NADH, FADH2, and ATP, which then goes on to produce even more energy for the cell (4).  Acetyl CoA is also very important to fermentation, where the acetyl group can be used to create Acetic acid, Isopropanol, Butanol, Butyric acid, or Ethanol (4). Thus, the conversion of acetate to Acetyl CoA is essential to cellular metabolism universally, so its regulation can induce a large effect on cells.  However, the addition of an acetyl group to the residue lys609 of Acs blocks the conversion to the Ac-AMP intermediate (7).  This acetylation, however, does not affect the formation of Ac-CoA from Ac-AMP (6).  The acetyl donor necessary for acetylation to occur comes form Ac-CoA itself, therefore, a feedback inhibition mechanism. 
In using this process to control Ac-CoA production and consequently energy production in the cell, Acs must be deacetylated to resume normal enzymatic function.  The proteins that perform this function are called sirtuins, which are nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases previously found in Eukaryotes and Archea to be important in chromosome stability and gene silencing (6).  Specifically, however, in the deacetylation of Acs, it is the CobB protein (encoded by the cobB gene) that is responsible for the reactivation of the metabolic enzyme in S. enterica (7).  CobB removes the inhibitory acetyl group from the inactive Acs, creating 2’-O-acetyl-ADPr, the functional protein (6). 
The gene encoding the protein acetyltransferase enzyme (Pat) responsible for the acetylation of Acs was recently identified in S. enterica (6).  Pat was determined as the acetyltransferase enzyme by V. J. Starai and J. C. Escalante-Semerena who isolated strains of S. enterica lacking the putative Pat enzyme to determine the bacteria’s ability to grow at low acetate concentrations (<10mM).  Tranposable elements were used to transform the cells and knockout different combinations of functions to determine their essential purpose in the regulation of cellular metabolism.  At the low acetate concentration, four different strains were grown: wild type, pta cobB (lacking cobB sirtuin), pta pat (lacking pat enzyme), and pta cobB pat (lacking cobB sirtuin and pat enzyme).  The pta pat and pta cobB pat strains grew at wild-type levels, and the growth of the pta cobB strain was greatly inhibited.  This showed that the Pat enzyme was in fact responsible for the acetylation of Acs (6).  This was evidenced because in each case when Pat was inactive, growth was normal, while growth was inhibited in the presence of Pat without the cobB sirtuin to reactivate enzymes, thus leaving them in their inactive states (6). 
The molecular interactions involved in the binding of Pat to Acs have been found to occur at a lysyl residue, Lys609 (6). The residue Lys609 was identified by using a time course experiment of deacetylation of Acs.  This method was based on the idea that if Acs was being acetylated at residue Lys906, the cobB sirtuin would be the deacetylation catalyst.  The Lys609 binding site was confirmed with the use of Matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry peptide fingerprinting by looking for the number and locus of acetyl groups transferred from Pat.  A high intensity signal was confirmed to represent the SGKAcIMR612 peptide in Acs where one acetyl group was observed (6).  Later studies also determined Leu641, another residue lying close to the acetylation site, as a vital amino acid for the acetylation of Acs to occur (7).  This was done by isolating unacetylated variants of Acs using a mutagenized plasmid and inhibiting acs genes (genes encoding acetyl-coenzyme A synthetase) and phosphotransacetylase genes.  A single base substitution was induced in the acs gene resulting in the placement of a prolyl residue where the leucyl residue normally resides.  Results showed that mutants were inviable with the addition of the propyl residue (7).  Mutations were induced changing the leucine to Gln and Ala to check for specificity of the leucine residue.  Results showed that Leu641 was necessary and was not specific to the propyl residue as initially tested (7). 
This precise protein regulation of the vital metabolic enzyme acetyl-coenzyme A (Ac-CoA) synthetase shows a critical and well-directed function of acetylation in in Salmonella enterica.


==Acetylation in Bacillus subtilis==
==Acetylation in Bacillus subtilis==

Revision as of 18:58, 15 April 2009

Introduction

The processes of transcription and translation are extremely complicated and require a diverse range of enzymatic proteins and cellular machinery to create the desired result, protein. It seems that the simple conversion of DNA to mRNA in transcription and the transformation of mRNA to amino acids is straightforward and continuous. However, the complicated mechanisms involved in creating proteins are intricately regulated at many steps throughout the process.

Transcription begins with the recruitment of RNA polymerase by sigma factors, which recognize the promoters of specific genes to be transcribed. Sigma factors present a very coordinated form of regulation before transcription has even begun. Sigma factors recognize consensus sequences of DNA (common sequences amongst promoters) and therefore can initiate the transcription of multiple proteins (4). Thus, transcription is carefully regulated by the presence or absence of pertinent sigma factors, which is controlled by sensory mechanisms within the cell that determine stress levels and deficiencies of certain nutrients. The accumulation of a certain sigma factor can cause the transcription of a specific DNA sequence whose gene products are necessary in the cell at that particular time. After sigma binding, the DNA is unwound, and RNA polymerase binds tightly to the DNA. The RNA polymerase then moves along the sequence of DNA, creating mRNA. Transcription is terminated after the stop codon is reached. Even here, the presence of GC rich sequences regulates transcriptional speed and can temporarily halt DNA transcription to allow for the ribosome to catch up in translation. These halts in transcription, either via binding to the protein Rho or the formation of a GC-rich loop, can cause termination if the termination signals are met with RNA polymerase (4).

Before the RNA sequence can be bound to the ribosome and translated, the ribosome itself must come together. This is initiated with the transcription of rRNA genes that are processed by RNases and configured into a secondary structure (4). Simulaneously, the ribosomal subunits come together with the rRNA forming the ribosome. With the necessity of transcribing rRNA genes and the presence of the ribosomal subunits, regulation can control whether the cell can and wants to perform translation. Protein elongation occurs when mRNA at the ribosome is met with tRNAs carrying the appropriate amino acid that is coded by the mRNA. Even tRNAs require correct pairing with amino acids before reaching the ribosome; this process is performed by aminoacyl-tRNA transferases (4). This enzyme could also be modified and hindered, thus regulating translation. The completion of successful protein synthesis is not complete without precise folding of the protein. The correct folding of proteins is crucial to their functioning within the cell. Many proteins are modified by removing the N-formyl group from the N-terminus, by adenylylation, by phosphorylation, or by acetylation, thus regulating their function by modification of the protein itself (4).

Regulation is occurring at frequent steps in this process of creating a functional protein. This regulation is of vital importance in most cells because of the colossal energy cost of transcription and translation. The formation and functioning of a ribosome alone can consume 40% of a bacterial cell’s energy, and therefore, the unnecessary production of proteins is extremely wasteful to a living cell (4). This limited energy must be conserved to carry out the numerous functions of the cell, and gene products must only be made when needed. Many stresses induce the need for certain gene products and suppress the need for others. For example, during starvation, many cells activate proteins that break down sugars for sustenance while others may activate ribosomal proteins that will transcribe necessary gene products. Other stresses, such as heat shock, intense pressure, and osmotic stress, may induce the formation or release of stability proteins to help the cell retain itself in extreme conditions. Stresses such as these can be sudden, and an immediate response may be necessary for the survival of the cell. The quickest method of regulation is post-translationally, or the modification of the proteins themselves. It is known that slight changes to the proteins can easily result in the fully inhibited functioning of the molecule. Therefore, as mentioned earlier, the simple addition of an acetyl group (acetylation) can inhibit the functioning of a protein or enzyme, thus controlling its ability to perform within the cell.

Acetylation and deacetylation was first determined in eukaryotes in the regulation of histones. Since, many other proteins have also been found to be acetylated, including proteins involved in DNA-protein interactions, transcription, and protein stability. Acetylation has also been found to have applications to age-related diseases, including cancer and cardiovascular diseases, for example (8).

Here, the post-translational effect of acetylation and deacetylation on proteins in bacterial cells is explored, both in regulating proteins involved in cellular processes such as metabolism and in proteins involved in translation itself. In Salmonella enterica, an aerobic, gram-negative bacterium, acetylation of Acetyl-CoA Synthetase (Acs) inhibits its funtion, which transforms Acetate to Acetyl-CoA (6). A similar mechanism in the gram-positive soil bacterium Bacillus subtilis is used to regulate Acs by way of an operon (1). Lastly, Escherichia coli, the gram-negative bacterium commonly found in mammalial intestines, has shown to have numerous sites of acetylation in various proteins, including those directly involved in transcription (8). The regulatory effect of these modifications is of extreme necessity to cell survival because of its rapid response in changing conditions. The grave importance and prevalence of these acetylation and deacetylation systems makes this form of regulation an emerging area of study because of its application to industry, the environment, and plant, animal, and human pathogens.

Acetylation in Salmonella enterica

[[Zbc0300513090002.jpeg⎮thumb⎮⎮1800px⎮⎮right⎮Acs proteins in S. enterica are acetylated by Pat, rendering them inactive. The sirtuin cobB deacetylates Acs with the use of NAD+. Once, deacetylated, acetate can be converted to the intermediate acetyl-AMP and then acetyl coenzyme A.]]

In the bacteria Salmonella enterica, post-translational modification of protein function has been determined to have a metabolic role by interacting with acetyl-coenzyme A (Ac-CoA) synthetase (Acs), the enzyme responsible for the conversion of acetate to the acetyl-adenosine monophosphate (Ac-AMP) intermediate and then to Ac-CoA (6). Acetyl-CoA is fundamental to cell metabolism, most importantly in the TCA cycle. Acetyl CoA is received mostly from the breakdown of pyruvate, but also from other catabolic pathways such as fat, amino acid, and benzoate breakdown. Acetyl CoA is taken up into the TCA cycle by incorporation via the combination of the acetyl group with oxaloacetyate. The cycle can then proceed to produce 3NADH, FADH2, and ATP, which then goes on to produce even more energy for the cell (4). Acetyl CoA is also very important to fermentation, where the acetyl group can be used to create Acetic acid, Isopropanol, Butanol, Butyric acid, or Ethanol (4). Thus, the conversion of acetate to Acetyl CoA is essential to cellular metabolism universally, so its regulation can induce a large effect on cells. However, the addition of an acetyl group to the residue lys609 of Acs blocks the conversion to the Ac-AMP intermediate (7). This acetylation, however, does not affect the formation of Ac-CoA from Ac-AMP (6). The acetyl donor necessary for acetylation to occur comes form Ac-CoA itself, therefore, a feedback inhibition mechanism.

In using this process to control Ac-CoA production and consequently energy production in the cell, Acs must be deacetylated to resume normal enzymatic function. The proteins that perform this function are called sirtuins, which are nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases previously found in Eukaryotes and Archea to be important in chromosome stability and gene silencing (6). Specifically, however, in the deacetylation of Acs, it is the CobB protein (encoded by the cobB gene) that is responsible for the reactivation of the metabolic enzyme in S. enterica (7). CobB removes the inhibitory acetyl group from the inactive Acs, creating 2’-O-acetyl-ADPr, the functional protein (6).

The gene encoding the protein acetyltransferase enzyme (Pat) responsible for the acetylation of Acs was recently identified in S. enterica (6). Pat was determined as the acetyltransferase enzyme by V. J. Starai and J. C. Escalante-Semerena who isolated strains of S. enterica lacking the putative Pat enzyme to determine the bacteria’s ability to grow at low acetate concentrations (<10mM). Tranposable elements were used to transform the cells and knockout different combinations of functions to determine their essential purpose in the regulation of cellular metabolism. At the low acetate concentration, four different strains were grown: wild type, pta cobB (lacking cobB sirtuin), pta pat (lacking pat enzyme), and pta cobB pat (lacking cobB sirtuin and pat enzyme). The pta pat and pta cobB pat strains grew at wild-type levels, and the growth of the pta cobB strain was greatly inhibited. This showed that the Pat enzyme was in fact responsible for the acetylation of Acs (6). This was evidenced because in each case when Pat was inactive, growth was normal, while growth was inhibited in the presence of Pat without the cobB sirtuin to reactivate enzymes, thus leaving them in their inactive states (6).

The molecular interactions involved in the binding of Pat to Acs have been found to occur at a lysyl residue, Lys609 (6). The residue Lys609 was identified by using a time course experiment of deacetylation of Acs. This method was based on the idea that if Acs was being acetylated at residue Lys906, the cobB sirtuin would be the deacetylation catalyst. The Lys609 binding site was confirmed with the use of Matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry peptide fingerprinting by looking for the number and locus of acetyl groups transferred from Pat. A high intensity signal was confirmed to represent the SGKAcIMR612 peptide in Acs where one acetyl group was observed (6). Later studies also determined Leu641, another residue lying close to the acetylation site, as a vital amino acid for the acetylation of Acs to occur (7). This was done by isolating unacetylated variants of Acs using a mutagenized plasmid and inhibiting acs genes (genes encoding acetyl-coenzyme A synthetase) and phosphotransacetylase genes. A single base substitution was induced in the acs gene resulting in the placement of a prolyl residue where the leucyl residue normally resides. Results showed that mutants were inviable with the addition of the propyl residue (7). Mutations were induced changing the leucine to Gln and Ala to check for specificity of the leucine residue. Results showed that Leu641 was necessary and was not specific to the propyl residue as initially tested (7).

This precise protein regulation of the vital metabolic enzyme acetyl-coenzyme A (Ac-CoA) synthetase shows a critical and well-directed function of acetylation in in Salmonella enterica.

Acetylation in Bacillus subtilis


Include some current research in each topic, with at least one figure showing data.

Acetylation in Escherichia coli


Include some current research in each topic, with at least one figure showing data.

Conclusion


Overall paper length should be 3,000 words, with at least 3 figures.

References

(1) Gardner, J. G., F. J. Grundy, T. M. Henkin, and J. C. Escalante-Semerena. August 2006. Control of Acetyl-Coenzyme A Synthetase (AcsA) Activity by Acetylation/Deacetylation without NAD+ Involvement in Bacillus subtilis. Journal of Bacteriology 188:5460-5468.

(2) Gardner, J. G. and J. C. Escalante-Semerena. July 2008. Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis. Journal of Bacteriology 190:5132-5136.

(3) Ramagopal, S. and A. R. Subramanian. May 1974. Alteration of the Acetylation Level of Ribosomal Protein L12 During Growth Cycle of Escherichia coli. Proc. Nat. Acad. Sci 71:2136-2140.

(4) Slonczewski, J. L., J. W. Foster, 2009, Microbiology: An Evolving Science: New York, W. W. Norton & Company, Inc. p. 257-300.

(5) Starai, V. J., H. Takahashi, J. D. Boeke, and J. C. Escalante-Semerena. 2004. A Link Between Transcription and Intermediary Metablism: A Role for Sir2 in the Control of Acetyl-Coenzyme A Synthetase. Current Opinion in Microbiology 7:115-119.

(6) Starai, V. J. and J. C. Escalante-Semerena. 2004. Identification of the Protein Acetyltransferase (Pat) Enzyme that Acetylates Acetyl-CoA Synthetase in Salmonella enterica. J. Mol. Biol 340:1005-1012.

(7) Starai, V. J., J. G. Gardner, and J. C. Escalante-Semerena. July 2005. Residue Leu-641 of Acetyl-CoA Synthetase is Critical for the Acetylation of Residue Lys-609 by the protein Acetyltransferase Enzyme of Salmonella enterica. The Journal of Biological Chemistry 280:26200-26205.

(8) Yu, B. J., J. A. Kim, J. H. Moon, S. E. Ryu, and J. Pan. January 2008. The Diversity of Lysine-Acetylated Proteins in Escherichia coli. J. Microbiol. Biotechnol. 18:1529-1536.