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1. Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1– tomato PTO immune complex via acetylation

   Jeleńska, J. ; Lee, J. ; Manning, A.J. ; Wolfgeher, D.J. ; Ahn, Y. ; Walters-Marrah, G. ; Lopez, I.E. ; Garcia, L. ; Sheri A. McClerklin, S.A. ; Michelmore, R.W. ; et al ( January 2021 , PLOS pathogens)

   null (Ed.)

   The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as avirulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass 44 spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues. 

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2. Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

   https://doi.org/10.1371/journal.ppat.1010017  

   Jeleńska, Joanna ; Lee, Jiyoung ; Manning, Andrew J ; Wolfgeher, Donald J ; Ahn, Youngjoo ; Walters-Marrah, George ; Lopez, Ivan E ; Garcia, Lissette ; McClerklin, Sheri A ; Michelmore, Richard W ; et al ( November 2021 , PLOS Pathogens)

   Shan, Libo (Ed.)

   The plant pathogenPseudomonas syringaesecretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector ofP.syringaepv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also thePsyeffectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and thePsyeffectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. InPsy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psyand the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psyfunction but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psyand PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.

    

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3. Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling

   https://doi.org/10.1002/cpz1.497  

   Song, Jiabao ; Han, Zhen ; Zheng, Y. George ( July 2022 , Current Protocols)

   Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC.

   Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin

   Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization

   Support Protocol 1: Expression and purification of HAT mutants

   Support Protocol 2: Synthesis of Ac‐CoA surrogates

   Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates

   Basic Protocol 3: Chemoproteomic identification of HAT substrates

   Basic Protocol 4: Validation of specific HAT substrates with western blotting

    

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4. Endogenous expression of inactive lysine deacetylases reveals deacetylation-dependent cellular mechanisms

   https://doi.org/10.1371/journal.pone.0291779  

   Toro, Tasha B. ; Skripnikova, Elena V. ; Bornes, Kiara E. ; Zhang, Kun ; Watt, Terry J. ( September 2023 , PLOS ONE)

   Li, Zhiming (Ed.)

   Acetylation of lysine residues is an important and common post-translational regulatory mechanism occurring on thousands of non-histone proteins. Lysine deacetylases (KDACs or HDACs) are a family of enzymes responsible for removing acetylation. To identify the biological mechanisms regulated by individual KDACs, we created HT1080 cell lines containing chromosomal point mutations, which endogenously express either KDAC6 or KDAC8 having single inactivated catalytic domain. Engineered HT1080 cells expressing inactive KDA6 or KDAC8 domains remained viable and exhibited enhanced acetylation on known substrate proteins. RNA-seq analysis revealed that many changes in gene expression were observed when KDACs were inactivated, and that these gene sets differed significantly from knockdown and knockout cell lines. Using GO ontology, we identified several critical biological processes associated specifically with catalytic activity and others attributable to non-catalytic interactions. Treatment of wild-type cells with KDAC-specific inhibitors Tubastatin A and PCI-34051 resulted in gene expression changes distinct from those of the engineered cell lines, validating this approach as a tool for evaluating in-cell inhibitor specificity and identifying off-target effects of KDAC inhibitors. Probing the functions of specific KDAC domains using these cell lines is not equivalent to doing so using previously existing methods and provides novel insight into the catalytic functions of individual KDACs by investigating the molecular and cellular changes upon genetic inactivation.

    

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5. The role of lysine acetylation in the function of mitochondrial ribosomal protein L12

   https://doi.org/10.1002/prot.26654  

   Paluch, Katelynn V. ; Platz, Karlie R. ; Rudisel, Emma J. ; Erdmann, Ryan R. ; Laurin, Taylor R. ; Dittenhafer‐Reed, Kristin E. ( December 2023 , Proteins: Structure, Function, and Bioinformatics)

   Mitochondria play a central role in energy production and cellular metabolism. Mitochondria contain their own small genome (mitochondrial DNA, mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The mitochondrial proteome, including the mitochondrial transcriptional machinery, is subject to post‐translational modifications (PTMs), including acetylation and phosphorylation. We set out to determine whether PTMs of proteins associated with mtDNA may provide a potential mechanism for the regulation of mitochondrial gene expression. Here, we focus on mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize mitochondrial RNA polymerase (POLRMT) and promote transcription. Numerous acetylation sites of MRPL12 were identified by mass spectrometry. We employed amino acid mimics of the acetylated (lysine to glutamine mutants) and deacetylated (lysine to arginine mutants) versions of MRPL12 to interrogate the role of lysine acetylation in transcription initiation in vitro and mitochondrial gene expression in HeLa cells. MRPL12 acetyl and deacetyl protein mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT. We analyzed mtDNA content and mitochondrial transcript levels in HeLa cells upon overexpression of acetyl and deacetyl mimics of MRPL12. Our results suggest that MRPL12 single‐site acetyl mimics do not change the mtDNA promoter binding ability of POLRMT or mtDNA content in HeLa cells. Individual acetyl mimics may have modest effects on mitochondrial transcript levels. We found that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 in vitro, suggesting a potential role for dynamic acetylation controlling MRPL12 function in a role outside of the regulation of gene expression.

    

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