Acetylation Of Actin Research Articles

Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish.

Actin is a critical component of the eukaryotic cytoskeleton. In animals, actins undergo unique N-terminal processing by dedicated enzymes resulting in mature acidic and acetylated forms. The final step, N-terminal acetylation, is catalyzed by NAA80 in humans. N-terminal acetylation of actin is crucial for maintaining normal cytoskeletal dynamics and cell motility in human cell lines. However, the physiological impact of actin N-terminal acetylation remains to be fully understood. We developed a zebrafish naa80 knockout model and demonstrated that Naa80 acetylates both muscle and non-muscle actins in vivo. Assays with purified Naa80 revealed a preference for acetylating actin N-termini. Zebrafish lacking actin N-terminal acetylation exhibited normal development, morphology, and behavior. In contrast, humans with pathogenic actin variants can present with hypotonia and hearing impairment. Whereas zebrafish lacking naa80 showed no obvious muscle defects or abnormalities, we observed abnormal inner ear development, small otoliths, and impaired response to sound. In conclusion, we have established that zebrafish Naa80 N-terminally acetylates actins in vitro and in vivo, and that actin N-terminal acetylation is essential for normal hearing.

Act88F pseudo-acetylation of lysine 50 (K50) negatively regulates muscle function in Drosophila

Non-histone post-translational modifications (PTMs) are an increasingly important area of study that contribute to our understanding for protein function in the control of health and disease. Protein PTMs, such as acetylation, are emerging as key regulators of striated muscle function, yet our understanding of how non-histone protein acetylation affects muscle physiology remains fragmentary. Previous reports from our lab identified lysine (K) 52 of skeletal muscle alpha actin (ACTA1) to be acetylated, yet no reports have shown a role for ACTA1 acetylation in muscle function. Act88F is 93% homologous to ACTA1 and K50 is identical to K52 of ACTA1. Thus, for these studies, we mutated K50 with a glutamine (Q) on the Act88F gene in the indirect flight muscle (IFM) of Drosophila to mimic acetylation. We examined physiological function (i.e. flight and climbing) as well as biophysical changes between acetylated Act88F and the associated myofibrillar proteins involved in contraction and relaxation (myosin, tropomyosin, and troponin). We report that Act88F K50Q pseudo-acetylation resulted in a flightless phenotype and decreased climbing ability. In addition, we report that Act88F K50Q flies had increased IFM damage as examined via fluorescent imaging. Interestingly, Act88F K50Q did not change actin-myosin binding, actin sliding velocity, or Ca2+ sensitivity in actin-motility assays. However, Act88F pseudo-acetylation did decrease actin filament length, but only after interacting with myosin, implying that Act88F acetylation increases actin filament breaking. These data suggest that Act88F acetylation at K50 worsens muscle function by destabilizing filamentous actin, resulting in muscle tearing and damage to the IFM, which results in loss of flight. This work suggests that actin acetylation, at least on lysine 50, in response to environmental stressors or diet could greatly change muscle function and sarcomere integrity. This research was funded by the Dennis Meiss & Janet Ralston Fund for Nutri-epigenetic Research, the National Institute for General Medical Sciences (NIGMS) of the NIH (P20 GM130459), the National Heart, Lung, and Blood Institute of the NIH (R15 HL143496), National Institute of Aging of the NIH (R21 AG077248) and NSF EPSCOR Track II (OIA-1826801) to B.S.F. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal muscle alpha actin (ACTA1) acetylation negatively regulates muscle function in response to obesity

Post‐translational modifications (PTMs) of proteins are an increasingly important area of study that contributes to our understanding for protein function in the control of health and disease. Acetylation, for example, has long been associated with histone modifications to control gene expression, yet emerging omics data demonstrates that acetylation occurs across the proteome. Our most recent work further shows global shifts in non‐histone protein acetylation within the left ventricle (LV) in response to diet‐induced obesity. Despite these advances in omics technologies, our understanding for how non‐histone protein acetylation regulates cardiac biology remains a ‘black box’. In this study, we examined the functional importance for skeletal muscle alpha actin (ACTA1) acetylation in the control of muscle function. For this, we used actin‐motility assays to examine the biophysical changes between acetylated ACTA1 and the other myofibrillar proteins involved in cardiac contraction and relaxation (e.g. myosin, tropomyosin). Here, we show that ACTA1 acetylation increased actin‐myosin binding, decreased actin sliding velocity and increased Ca2+ sensitivity. These data suggest that ACTA1 acetylation significantly alters muscle function. Of note, we previously published that lysine (K) residue 50 (K50) of ACTA1 was acetylated, similar to other proteomics reports. Interestingly, the positively charged K50 on ACTA1 sits juxtaposed to the negatively charged aspartic acid (D) residue 391 of the cardiac myosin binding protein (cMy‐BP). cMy‐BP is essential for actin‐myosin interactions, and this suggests an important role for ACTA1 K50 acetylation in the control of muscle contraction. Thus, we knocked out endogenous actin (Act88F) from the indirect flight muscles (IFMs) of D. melanogaster and inserted an Act88F K50Q mutant, which mimics acetylation specifically at this site. Previous reports have shown that IFMs behave similarly to cardiac muscle. Phenotypically, the K50Q flies behave similar to their wild‐type counterparts. However, current studies are underway to examine changes in flight as well as purify the K50Q actin for actin‐motility assays to examine biophysical changes in myofibrillar proteins. Combined, our data suggests that ACTA1 acetylation negatively impacts muscle function in response to obesity, current work will determine the implications for K50 actin acetylation in this phenotype.

NAA80 bi-allelic missense variants result in high-frequency hearing loss, muscle weakness and developmental delay.

The recent identification of NAA80/NAT6 as the enzyme that acetylates actins generated new insight into the process of post-translational actin modifications; however, the role of NAA80 in human physiology and pathology has not been clarified yet. We report two individuals from a single family harbouring a homozygous c.389T>C, p.(Leu130Pro) NAA80 genetic variant. Both individuals show progressive high-frequency sensorineural hearing loss, craniofacial dysmorphisms, developmental delay and mild proximal and axial muscle weakness. Based on the molecular structure, we predicted and confirmed the NAA80 c.389T>C, p.(Leu130Pro) variant to result in protein destabilization, causing severely decreased NAA80 protein availability. Concurrently, individuals exhibited a ∼50% decrease of actin acetylation. NAA80 individual derived fibroblasts and peripheral blood mononuclear cells showed increased migration, increased filopodia counts and increased levels of polymerized actin, in agreement with previous observations in NAA80 knock-out cells. Furthermore, the significant clinical overlap between NAA80 individuals and individuals with pathogenic variants in several actin subtypes reflects the general importance of controlled actin dynamics for the inner ear, brain and muscle. Taken together, we describe a new syndrome, caused by NAA80 genetic variants leading to decreased actin acetylation and disrupted associated molecular functions. Our work suggests a crucial role for NAA80-mediated actin dynamics in neuronal health, muscle health and hearing.

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys326 and Lys328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin-Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin-Tpm motility that persisted in the presence of Tn and submaximal Ca2+ Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys326 and Lys328, also resulted in less inhibited F-actin-Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn-Tpm-based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys328, modulates muscle contraction via disrupting inhibitory Tpm positioning.

Lysine acetylation of cytoskeletal proteins: Emergence of an actin code.

Reversible lysine acetylation of nuclear proteins such as histones is a long-established important regulatory mechanism for chromatin remodeling and transcription. In the cytoplasm, acetylation of a number of cytoskeletal proteins, including tubulin, cortactin, and the formin mDia2, regulates both cytoskeletal assembly and stability. More recently, acetylation of actin itself was revealed to regulate cytoplasmic actin polymerization through the formin INF2, with downstream effects on ER-to-mitochondrial calcium transfer, mitochondrial fission, and vesicle transport. This finding raises the possibility that actin acetylation, along with other post-translational modifications to actin, might constitute an "actin code," similar to the "histone code" or "tubulin code," controlling functional shifts to these central cellular proteins. Given the multiple roles of actin in nuclear functions, its modifications might also have important roles in gene expression.

N-terminal acetylation of actin by NAA80 is essential for structural integrity of the Golgi apparatus

N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization.

Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.

Quantification of intracellular N-terminal \u03b2-actin arginylation

Actin is a ubiquitous, essential, and highly abundant protein in all eukaryotic cells that performs key roles in contractility, adhesion, migration, and leading edge dynamics. The two non-muscle actins, β- and γ-, are ubiquitously present in every cell type and are nearly identical to each other at the amino acid level, but play distinct intracellular roles. The mechanisms regulating this distinction have been the focus of recent interest in the field. Work from our lab has previously shown that β-, but not γ-, actin undergoes N-terminal arginylation on Asp3. While functional evidence suggest that this arginylation may be important to actin’s function, progress in these studies so far has been hindered by difficulties in arginylated actin detection, precluding estimations of the abundance of arginylated actin in cells, and its occurrence in different tissues and cell types. The present study represents the first antibody-based quantification of the percentage of arginylated actin in migratory non-muscle cells under different physiological conditions, as well as in different cells and tissues. We find that while the steady-state level of arginylated actin is relatively low, it is consistently present in vivo, and is somewhat more prominent in migratory cells. Inhibition of N-terminal actin acetylation dramatically increases the intracellular actin arginylation level, suggesting that these two modifications may directly compete in vivo. These findings constitute an essential step in our understanding of actin regulation by arginylation, and in uncovering the dynamic interplay of actin’s N-terminal modifications in vivo.

Calreticulin stabilizes F-actin by acetylating actin and protects microvascular endothelial cells against microwave radiation

AimsCalreticulin (CRT) is a multifunctional protein that protects endothelial cells by alleviating actin cytoskeleton injury, but the underlying mechanism remains unclear. CRT was recently identified as a novel acyltransferase; acetylation at the N-terminus of actin monomers strengthens actin polymerization. This study was undertaken to determine whether CRT protects human microvascular endothelial cells (HMECs) against microwave radiation through actin acetylation. Materials and methodsWe prepared a eukaryotic-derived recombinant CRT and incubated the HMECs with it prior to microwave exposure. We then assessed cell injury and endothelial function, detected actin polymerization and acetylation after HMECs exposure to S-band high-power microwaves. Coimmunoprecipitation, pull-down, and ex vitro acetylation reaction were performed to determine whether actin is a novel substrate of CRT acyltransferase. Finally, we employed the mutant experiments to demonstrate the acetylation sites contributing to CRT acetyltransferase activity. Key findingsMicrowave radiation induced severe cell injury and endothelial contact dysfunction, reduced the polymerization of actin filaments, and destroyed the actin arrangement, ultimately reducing acetylated actin expression. CRT treatment upregulated actin acetylation levels, promoted polymerization, and facilitated thicker and longer F-actin stress fibre formation. Pre-incubation with CRT rescued microwave-induced cell injury, decreased actin acetylation, and rendered the actin cytoskeleton radiation-retardant. The level of acetyl-actin was positively correlated with actin polymerization. Actin was identified as a novel substrate of CRT, being acetylated mainly through the CRT P-domain at lys-206 and -207. SignificanceThis work provides a better understanding of the underlying mechanism of CRT-induced cytoprotection, and suggests a novel therapeutic target for microwave radiation-related diseases with endothelial dysfunction.

Acetylation of Actin K328 Contributes to a Loss in Tropomyosin-Mediated Inhibition of Myosin Binding

Lysine deacetylases (KDAC's) catalyze the removal of acetyl groups from lysine side chains, while lysine acetylases (KAT's) perform the reverse reaction. KDAC inhibition was shown to increase lysine acetylation of muscle proteins, and was associated with enhanced Ca2+-sensitivity in skinned fibers and increased relaxation rate in isolated myofibrils. Interestingly, the KAT enzyme, PCAF, directly acetylates myosin in vitro, and actin has been implicated as a potential target of PCAF. Therefore, we attempted to acetylate actin with PCAF, in vitro, to determine its potential effect(s) on muscle function. PCAF underwent autoacetylation and bound to actin, yet failed to acetylate it. Chemical treatment of actin with acetic anhydride, however, increased lysine acetylation roughly 200-fold. Surprisingly, in vitro motility (IVM) of (100 μg/ml myosin concentration) and tropomyosin affinity for acetylated F-actin were indistinguishable from unacetylated control. However, IVM of acetylated F-actin, performed at low myosin concentration (12.5 μg/ml) and in the presence of tropomyosin, revealed a significant 63% increase in motile filaments relative to unacetylated control (p < 0.0001), suggesting that actin acetylation reduces tropomyosin-mediated inhibition of myosin binding. Since K326 and K328 have been reported as the most reactive lysines on actin, are acetylated in vivo, and pseudo-acetylation of both modulates muscle contraction, we assessed the effect of K326Q and K328Q pseudo-acetylated actin on Ca2+-mediated thin filament activation via regulated IVM. Although reconstituted thin filaments containing K326Q (pCa50 = 6.36 ± 0.03) did not alter Ca2+-sensitivity relative to WT (pCa50 = 6.37 ± 0.03), K328Q induced a significant leftward shift in Ca2+-sensitivity (ΔpCa50 = 0.19; p < 0.0001), consistent with a loss in tropomyosin-mediated inhibition. The data indicate that actin K328 acetylation modulates contractile function, which makes it a potential mechanism for regulating muscle performance in vivo.

Actin polymerization and cell motility are affected by NAA80-mediated posttranslational N-terminal acetylation of actin

ABSTRACTActin is the most abundant protein in our cells, and also one of the most studied. Nevertheless, an important modifier of actin, the N-terminal acetyltransferase (NAT) for actin, remained unknown until now. The recent identification of the enzyme that catalyzes actin acetylation, has opened up for functional studies of unacetylated actin using knockout cells. This enzyme, called NAA80 (Nα-acetyltransferase 80) or NatH, belongs to the NAT family of enzymes, which together provides N-terminal acetylation for around 80 % of the human proteome. In many cases, N-terminal acetylation is essential. In the case of actin, the acetyl group that NAA80 attaches to actin plays an important role in actin’s polymerization properties as well as in actin’s function in cell migration.

Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization.

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100mW/cm2, for 6min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

Lysine acetyltransfer supports platelet function

The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function. Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of the identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments showed that platelets express p300, which is activated in an Src kinase-dependent manner upon platelet stimulation with the platelet glycoproteinVI agonist collagen-related peptide (CRP). Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin, and cortactin, as well as F-actin polymerization, integrin activation, and platelet aggregation. Super-resolution visualization of platelet actin-rich adhesion structures revealed abundant acK protein colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodium formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiologic role for lysine acetyltransferase activity in platelet function. Together, our findings reveal lysine acetyltransfer to be a potential regulator of platelet actin dynamics, and potential roles for lysine acetylation in the molecular coordination of platelet activation and function.

Nat3p and Mdm20p Are Required for Function of Yeast NatB Nα-terminal Acetyltransferase and of Actin and Tropomyosin

NatB Nalpha-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with Met-Glu- or Met-Asp- termini and subclasses of proteins with Met-Asn- and Met-Met- termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-Delta and mdm20-Delta mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNA-damaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.

Actin acetylation in Drosophila tissue culture cells.

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.