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Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. The 10 most studied PTMs. Main PTM databases. Computational methods for predicting PTMs. Tools for PTM prediction. Post-translational modifications in proteins: resources, tools and prediction methods.
Shahin Ramazi , Shahin Ramazi. Oxford Academic. Javad Zahiri. Revision received:. Editorial decision:. Select Format Select format. Permissions Icon Permissions. Abstract Posttranslational modifications PTMs refer to amino acid side chain modification in some proteins after their biosynthesis. Open in new tab Download slide. Table 1. Major databases for PTMs. General statistics. Number of covered organisms. Number of PTM types. Number of PTMs a.
Type of data and database b. Open in new tab. Table 2. Confusion matrix for PTM prediction tools. Experimentally validated PTMs. No PTM. Evaluation of post-translational modifications in histone proteins: a review on histone modification defects in developmental and neurological disorders.
Google Scholar Crossref. Search ADS. Recent progress in predicting posttranslational modification sites in proteins. Protein post-translational modifications and regulation of pluripotency in human stem cells. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Effect of posttranslational modifications on enzyme function and assembly.
Glycosylation site prediction using ensembles of Support Vector Machine classifiers. Small changes huge impact: the role of protein posttranslational modifications in cellular homeostasis and disease. Del Monte. Protein post-translational modifications and misfolding: new concepts in heart failure. However, because of the numerous cell activities that are directly governed by proteins, there is no doubt that the regulation of protein components in hPSCs should have profound influences on cellular pluripotency and differentiation capacity.
The consequences of altering proteins at the post-translational level in hPSCs are thus interesting issues applicable to the regulation of pluripotency. Advances in molecular biology and protein biochemistry have led to the development of several modern technologies to better examine the expression, post-translational modification and functional alteration of proteins at single-protein and proteomic levels 16 , 17 , 18 , 19 , 20 , Discoveries based on these methods have shed light on the importance of many PTMs in controlling protein functions, signaling networks and cell fates in hPSCs.
It is well known that protein glycosylation plays a critical role in the regulation of protein structure 22 , signal transduction 23 , cell-cell and cell-environment interactions 24 , 25 , 26 , immune responses 27 , 28 , hormone action 29 , cancer progression 30 and embryonic development 31 , In the glycosylation process, carbohydrate units can be covalently linked to proteins and edited through various biochemical reactions that are catalyzed by glycosyltransferases GTs and glycosidases in the endoplasmic reticulum ER and Golgi apparatus Figure 3.
There are four major types of protein glycosylation in mammalian cells: N -linked glycosylation, O -linked glycosylation, C -linked mannosylation and glypiation Among these types of protein glycosylation, N -linked and O -linked glycomodifications are the most abundant in cells. N -linked glycosylation often occurs on a large variety of nascent proteins.
O -linked monosaccharide modification of N-acetylglucosamine GlcNAc on serine, threonine or amino acid residues in close proximity to tyrosine phosphorylation sites is frequently observed in many cells. At these sites, glycosylation may contribute to the regulation of signaling pathways through a direct competition with serine and threonine phosphorylation or by indirectly perturbing the phosphorylation of tyrosine N -linked and O -linked protein glycosylation occurs in the ER and Golgi apparatus.
The synthesis of precursor glycans mannose-rich glycans begins on the cytosolic face of the ER and is completed after the glycans are flipped into the ER lumen and further branched by adding more units of mannose and glucose. ER-resident glycosyltransferases GTs transfer the precursor glycans to asparagine residues on nascent proteins to form N -linked precursor glycans.
When the proteins are transported into the Golgi apparatus, the N -linked precursor glycans are edited by Golgi-resident glycosidases GSs and GTs to form mature and structurally-diverse N -linked glycans. O -linked protein glycosylation is mainly performed by Golgi-resident GTs. Multiple lines of evidence support the importance of protein glycosylation and its potential role in the regulation of cellular pluripotency and differentiation of hPSCs. Many pluripotency-associated antigens e. Despite the relatively limited sample numbers and types of hPSCs, several studies using mass spectrometry 39 , 40 to analyze the glycan components of glycoproteins isolated from hPSCs and differentiated cells demonstrated that protein glycosylation differs considerably between pluripotent and non-pluripotent cells.
More recently, several studies have used lectin microarrays and transcriptomic analysis to perform large-scale, high-throughput characterization of protein glycosylation and glycosyltransferase expression in various types of undifferentiated hPSCs and differentiated cells.
These studies have provided definitive evidence showing significant differences between the glycomic fingerprints associated with these distinct cellular states 46 , 47 , 48 and led to the identification of a lectin biomarker that can be used to isolate viable hPSCs Regardless of the different methods used in these studies, their results appear to be in agreement with the idea that two types of glycomodifications, fucosylation and sialylation, are typically altered when hPSCs lose their pluripotency 39 , 40 , 46 , 48 , This suggests that these two types of protein glycosylation may be involved in the regulatory mechanisms underlying cellular pluripotency and lineage specification.
In support of this idea, many studies have demonstrated that fucosylation and sialylation are crucial for normal embryonic development and cell maturation, and that deficiencies in these glycomodifications can lead to the impairment of embryogenesis and somatic stem cell differentiation in mammalians and other vertebrates 31 , 32 , 50 , 51 , Moreover, certain fucosyltransferases and sialyltransferases are preferentially expressed in hPSCs 46 , 48 , suggesting a role for these enzymes in maintenance of the pluripotency-associated profile.
Although the mechanisms by which these glycosyltransferases may participate in the regulation of pluripotency and differentiation of hPSCs have not been well characterized, it is likely that their enzymatic activity orchestrates the functions of many pluripotency-related signaling molecules. A recent report published by Jang et al.
Although a similar regulatory mechanism has not yet been examined in hPSCs, this study demonstrated a direct link between protein glycosylation and pluripotency regulation that is highly likely to exist in human cells as well. Interestingly, O -GlcNAc transferase Ogt has also been identified as a stable binding partner for 5-methylcytosine hydroxylases Tet1 and Tet2 in mESCs, indicating that the protein glycosylation activity of Ogt may participate in the regulation of CpG island methylation and thus gene expression These studies also suggested that it may be possible to manipulate pluripotency in mammalian cells for research or clinical applications by controlling protein glycosylation.
Many mitogens and morphogens play important roles in the establishment and maintenance of cellular pluripotency in hPSCs in vitro. Additionally, there are numerous growth factors and cytokines involved in the optimization of signaling circuits during cell lineage specification and normal embryonic development. Like FGF2, other signaling factors such as Notch, Wnt proteins and epidermal growth factors EGFs , are intimately involved in the determination of stem cell fate 59 , The activities of these key regulators of cell differentiation and their associated signaling pathways are also influenced by their own glycosylation state and extracellular HSPGs 51 , 61 , 62 , 63 , Indeed, defects in protein glycosylation machinery frequently lead to the impairment of developmental signaling, the retardation of embryogenesis in animal models and the pathogenesis of human congenital disorders 31 , 32 , 50 , 51 , 52 , 62 , 65 , 66 , 67 , 68 , 69 , highlighting alternative mechanisms by which protein glycosylation may regulate pluripotent and differentiated states in hPSCs.
Although the potential mechanisms and functional significance of protein glycosylation in the regulation of cellular pluripotency in hPSCs require further exploration, the possible utility of unique glycosylation profiles in hPSCs has been appreciated and exploited in relevant fields.
To ensure the safety of cells differentiated from hPSCs for cell-based therapy, it is critical to remove residual undifferentiated hPSCs that are potentially tumorigenic. Moreover, to enhance the reproducibility and efficiency of differentiation methods, it may be desirable to select homogeneous undifferentiated hPSC populations in which all the cells have similar capacities and responses to differentiation stimuli.
Differential surface glycosylation features between hPSCs and non-pluripotent cells have been used to develop methods to remove undifferentiated cells and purify differentiated cell types 48 , Protein glycosylation marks in hPSCs can thereby be considered potential targets for developing a rigorous strategy for the quality control of hPSCs and their differentiated derivatives.
Similar to protein glycosylation, protein phosphorylation is involved in the regulation of a broad spectrum of cellular processes and states. The phosphorylation state of proteins in typical eukaryotic cells is mainly determined by the activity of protein kinases and phosphatases on their substrates.
Many kinases and phosphatases are also phosphorylation substrates, thereby forming mutually-dependent and hierarchically-regulated signaling loops and cascades Cell fate determination in hPSCs strongly depends on the balance between pluripotency and differentiation signalings. As shown in Figure 4 , many signaling pathways critically involved in the embryonic development and the modulation of gene expression for cellular pluripotency and differentiation are initiated from the activation of growth factor receptors that are known receptor tyrosine kinases RTKs; e.
It is notable that these signaling pathways have frequent crosstalk with each other, and that the steady state of cellular pluripotency is established on top of an intricate and yet delicately-balanced molecular interaction network 73 , Cell signaling pathways governed by protein phosphorylation and critically involved in embryonic development and the regulation of pluripotent states in PSCs.
Many growth factor receptors on the cell surface are receptor kinases. Upon ligand binding, these receptor kinases are fully activated and phosphorylate downstream, intracellular kinases to initiate phosphorylation signaling cascades that frequently regulate the translocation and activity of several transcription factors e.
These signal transduction pathways are highly interactive with each other and influenced by other proteins e. Several protein phosphatases e. In the stem cell field, many efforts are made to dissect the signaling networks regulated by protein phosphorylation in hPSCs and understand how they function as a whole to regulate cellular pluripotency and differentiation.
Advances in protein mass spectrometry have enabled the global, quantitative analysis of dynamic changes in phosphorylated proteins in cells. Several recent studies used phosphoproteomic approaches to systematically investigate phosphorylated proteins in hPSCs. The study done by Swaney et al.
Van Hoof et al. Moreover, the comparison between the proteomes and phosphoproteomes of a small number of hESCs and hiPSCs revealed functionally-associated differences in protein expression and phosphorylation in these two types of hPSCs, possibly related to residual regulatory characteristics of the somatic cells used for generating the hiPSCs It is therefore plausible that the protein phosphorylation modulates pluripotency in hPSCs by acting on the key factors, which are essential for pluripotency in addition to numerous signal transduction molecules.
Variations in protein expression and the phosphorylation state of different hPSC lines may affect their responses to environmental stimuli. Like glycoproteins, phosphoproteins appear to convey information regarding the pluripotent state of hPSCs. However, it is likely that the phosphoproteome or a subset of phosphoproteins could provide a sensitive and useful biomarker for monitoring pluripotency and differentiation in hPSCs. It is clear that both kinases and phosphatases play critical roles in the proper operation of cell signaling mediated by protein phosphorylation.
Unlike many kinases that have been well studied in somatic cells and hPSCs, the importance of protein phosphatases in the regulation of cellular pluripotency is less appreciated.
Despite the overwhelming amount of attention that has been focused on kinases in mammalian PSCs, protein phosphatases alkaline phosphatase in particular remain one of the earliest-discovered and most commonly used biomarkers for cellular pluripotency 78 , 79 , indicating the potential functional significance of protein phosphatases in PSCs.
Indeed, emerging data have shown that several phosphatases e. Moreover, suppression of these protein phosphatases inhibits hPSC exit from the pluripotent state during differentiation 80 , 81 , These studies also illustrate how phosphatases affect cellular pluripotency by altering protein phosphorylation in various signaling pathways, and establish a strong rationale for the development of a strategy to stabilize pluripotency by specific interference with the activity of certain phosphatases.
Numerous studies have suggested that the expression and activity of many protein kinases and phosphatases can be influenced by single nucleotide polymorphisms SNPs or rare point mutations existing in human genomes. These genetic variations are functionally associated with the differential regulation of signal transduction and the unequal susceptibility to a variety of disorders among different individuals 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , Although correlations between the differentiation capacity of hPSCs and these genetic variations have not been systematically examined, it is feasible that cellular pluripotency and differentiation propensity may differ in different hPSC lines partially due to intrinsic genetic variation that alters cell signaling mediated by protein phosphorylation.
As mentioned earlier, protein glycosylation and extracellular proteoglycans are critical for modulating growth factors and plasma membrane-bound receptor kinases to which they bind Figure 4. This suggests that protein glycosylation and phosphorylation are highly interactive in hPSCs, and that the perturbation of glycomodifications or glycoprotein expression on the cell surface may be frequently accompanied by drastic changes in phosphorylation signaling networks and the pluripotent state.
As shown in Figure 5 , two types of key regulators, histone acetyltransferases HATs and histone deacetylases HDACs , dynamically control the acetylation state of histones. The antagonistic actions of these enzymes on histones serve as an important mechanism for the epigenetic regulation of gene expression There are numerous examples showing that the acetylation state of proteins is highly relevant to their stability and activity in cells.
Defects in protein acetylation frequently result in severe abnormalities of development and physiology due to the dysregulation of gene expression and protein function in animal models, and are pertinent to the pathogenesis of many human diseases 95 , 96 , In addition, undifferentiated mESCs appear to have a higher level of global histone acetylation with transcriptional hyperactivity as compared to their differentiated derivatives These observations not only suggest the importance of protein acetylation in controlling cellular state and differentiation capacity, but also rationalize approaches to potentially correct these abnormalities or treat diseases by targeting HATs and HDACs.
The antagonistic actions of HATs and HDACs are required for regulating the acetylation of histone and non-histone proteins in many types of mammalian cells, including mouse and human PSCs. The acetylation state of histones affects chromatin structure and dictates the accessibility of promoter regions to the transcriptional machinery and the activation of gene expression.
In the pluripotent state, cells appear to have higher levels of global histone acetylation and chromatin accessibility for transcriptional machinery. Efforts studying HDACs as therapeutic targets in malignant cells have led to the development of a series of small-molecule inhibitors, particularly inhibitors of class I and II HDACs, that block their ability to catalyze protein deacetylation One of the most well-known HDAC inhibitors is suberoylanilide hydroxamic acid SAHA, Vorinostat , currently used as an anticancer therapeutic agent to treat patients with cutaneous T-cell lymphoma.
Interestingly, many HDAC inhibitors, including SAHA, valproic acid VPA , trichostatin A TSA and sodium butyrate, are known to significantly enhance the efficiency of reprogramming mouse or human somatic cells into iPSCs 99 , , , , suggesting that suppression of HDAC activity and regulation of protein acetylation is important for the establishment and modulation of cellular pluripotency.
Despite these reported discrepancies between mouse and human somatic cells, it is generally agreed that treatment of somatic cells with HDAC inhibitors during reprogramming can induce changes that drive cells toward the pluripotent state due to increased histone acetylation and activation of gene expression.
Two recent studies showed that the nucleosome remodeling and deacetylation NuRD complex containing HDAC1 and HDAC2 is functionally associated with suppression of pluripotency-associated gene expression and promotion of lineage commitment in mESCs , These studies reiterate the significant role of HDACs in the regulation of cellular pluripotency and suggest that HDACs regulate pluripotency through a mechanism involving chromatin remodeling.
It has been shown that many deacetylases are localized to the cytoplasm or frequently shuttle between the cytoplasm and nucleus This cytoplasmic localization provides these enzymes with the opportunity to interact with many non-histone proteins to modulate their acetylation state and function. Many of these non-histone substrates e.
In fact, several reports have indicated the importance of HDAC-mediated deacetylation of non-histone proteins for lineage specification and normal differentiation of particular cell types from stem cells , , Jain et al.
As HDAC inhibitors e. This may occur through modulating the post-translational regulation of TP53 and other signaling molecules that are relevant to embryogenesis or pluripotency, in addition to altering chromatin structures and gene expression. SIRT1 knockdown in hESCs or knockout in mESCs appears to have negligible effects on the expression of pluripotency factors prior to the induction of differentiation , In addition, hESCs with SIRT1 knockdown show greater changes in the expression of pluripotency-related and differentiation-related genes in response to differentiation cues, compared to wild-type hESCs Interestingly, Sirt1 deficiency impedes the downregulation of pluripotency factors, delays the induction of differentiation factors, and compromises hematopoietic lineage capacity in mESCs undergoing differentiation Although more studies are needed to comprehensively understand how SIRT1 exerts its function on the determination of cell fate in different types of PSCs, these findings indicate that the regulation of histone acetylation by sirtuins may greatly influence differentiation programs and lineage commitment.
A more recent study reveals that SIRT1 is elevated during cell reprogramming and facilitates the generation of miPSCs partially through the negative regulation of Tp53 acetylation and transcriptional activity , suggesting that the SIRT1-mediated protein deacetylation of non-histone substrates may play an important role in the establishment and maintenance of cellular pluripotency in hPSCs.
The deacetylation and modulation of different non-histone proteins by sirtuins during cell differentiation also provides a possible explanation for the perplexing and somewhat contradictory observations of phenotypic alterations induced by SIRT1 deficiency in hESCs and mESCs. Indeed, all the sirtuins have been shown to interact with non-histone substrates , which may vary in amount and composition in different cells.
Given the fact that most HDAC inhibitors can target multiple members within the HDAC family, the identification of any HDAC s and its substrate s fundamentally associated with pluripotency or differentiation remains an important and challenging task.
In addition, the significance of HATs and their mechanism of action with regard to the regulation of cellular pluripotency in hPSCs could be equally interesting. In addition, the conditional deletion of Trrap depletes the hematopoietic stem cell pool in mice These findings highlight the need to examine HATs in hPSCs to understand their potential roles in the regulation of pluripotency in human cells. Like the crosstalk between glycosylation and phosphorylation of proteins, there are many identified interactions between protein acetylation and phosphorylation signaling.
These potential regulatory interactions not only add another layer of complexity to the molecular mechanisms underlying cellular pluripotency regulated by protein acetylation, but also remind us that the treatment with HDAC inhibitors that selectively inhibit different types of HDACs may lead to distinct consequences in cellular reprogramming or differentiation of hPSCs.
The identity of the enzymes causing protein methylation remained unknown until the heterogeneous nuclear ribonucleoprotein hnRNP methyltransferase 1 HMT1, also known as RMT1 was first discovered in Saccharomyces cerevisiae less than 20 years ago Since then, numerous types of protein methyltransferases and their orthologs have been identified in yeast, fruit flies and mammals , It is now clear that protein methylation has profound influences on many biological events and that defects in protein methyltransferases may lead to severe phenotypic abnormalities during embryogenesis , Two types of protein methylation, arginine and lysine methylation Figure 6 , and their relevant methyltransferases have been frequently described.
There are 10 members in the protein arginine methyltransferase PRMT family and more than 30 members in the protein lysine methyltransferase PKMT family expressed by mammalian cells , Unlike acetylated lysine residues on histones, which are generally associated with the activation of gene expression, the methylation of different lysine residues on histones may lead to either activation or suppression of gene expression.
The biochemical reactions of arginine and lysine methylation are catalyzed by protein arginine methyltransferases PRMTs and protein lysine methyltransferases PKMTs in cells.
Depending on the number of methyl groups and types of methyltransferases that are involved in methylation, the reactions can result in the production of monomethylarginine, symmetric dimethylarginine, asymmetric dimethylarginine, monomethylated, dimethylated or trimethylated lysine in peptides. It has been reported that the methylation of histone H3 lysine 4 H3K4 , H3K36 and H3K79 is associated with active gene expression , , , and that the methylation of histone H3K9, H3K27 and H4K20 is involved in gene silencing , , , Due to its functional impact on transcription, it is foreseeable that histone methylation may participate in the regulation of pluripotent states by modulating the expression of pluripotency factors.
In fact, several reports have described genome-wide histone methylation patterns in hESCs and the dynamic changes in bivalent chromatin marks e. Many recent studies further examined correlations between gene expression and the methylation states of H3K4 and H3K27 in hESCs committing to the specific cell lineages and hiPSCs reprogrammed from somatic cells , , These studies suggest that histone methylation and chromatin remodeling are critical for the cardiac and pancreatic differentiation and are highly similar between hESCs and hiPSCs.
Furthermore, the data obtained from murine systems also demonstrated an essential role of Carm1 coactivator-associated arginine methyltransferase 1 -mediated histone arginine methylation in the regulation of cellular pluripotency in mESCs , and revealed the importance of histone lysine methylation for balancing quiescent and active states of hair follicle stem cells in vivo An illustration of amino acid residues where acetylation, methylation and phosphorylation frequently occur in core histones.
Certain lysines e. Trimethylation of H3K4 and H3K27 gives rise to the bivalent chromatin marks found at the transcription start sites of many genes involved in lineage commitment and development in undifferentiated hPSCs. The majority of these bivalent marks resolve into one or the other at each particular gene during cell differentiation, depending on the expression state of the genes in differentiated derivatives. In general, genes induced during the differentiation of hPSCs retain the H3K4 methylation marks, and genes silenced during differentiation retain the H3K27 methylation marks.
The phosphorylation of amino acid residues e. Being one of the additive PTMs, protein methylation is theoretically reversible and under the control of antagonistic reactions catalyzed by protein methyltransferases and demethylases. Unlike the intense attention paid to deacetylases in protein acetylation research, protein demethylases seem to receive much less attention than methyltransferases from researchers in relevant fields. Newly synthesized vRNPs are exported out from the nucleus through the nuclear pore complex, which relies on host cellular export machinery.
CRM1 is the major exporting factor responsible for the transport of influenza virus vRNPs from the nucleus to the cytoplasm. During this process, M1 plays an important role in facilitating the interaction between vRNPs and the host cellular export system.
M1 with phosphorylation modification plays a concerted role in promoting the nuclear export of vRNPs during influenza virus infection Bui et al.
Phosphorylation of M1 Y is essential for influenza virus replication, which controls the nuclear import of M1 and the subsequent nuclear export of progeny vRNPs by regulating the interaction between M1 and the nuclear import factor importin-1 Wang et al.
Phosphorylation of NP Y obstructs the nuclear export of vRNPs complexes and subsequently impairs virus assembly and polymerase activity through inhibiting the binding affinity of NP to export factor CRM1 Zheng et al. RanBP3 is a Ran-interacting protein that acts as a nuclear export cofactor of CRM1-mediated cargo during influenza virus infection.
The actin and microtubule cytoskeleton also play a critical role in viral replication Radtke et al. Actin cytoskeleton contraction and relaxation are predominantly regulated by phosphorylation and dephosphorylation of the regulatory subunit of MLC Lincoln, Influenza virus infection activates a series of signaling pathways that induce MLC phosphorylation and actin cytoskeleton remodeling. The inhibition of MLC phosphorylation leads to nuclear retention of influenza vRNPs complexes and a reduction of influenza virus replication, whereas the induction of MLC phosphorylation reverses the inhibitory effects of unphosphorylated MLC on the nuclear translocation of influenza virus vRNPs complexes Haidari et al.
The NP protein has an intrinsic ability to be exported from the nucleus to the cytoplasm, and the nuclear export ability of NP is partially regulated by phosphorylation. Influenza virus assembles at the host cell plasma membrane. Before budding, the newly synthesized vRNPs and viral membrane proteins HA, NA, and M2 are translocated to the budding site at the plasma membrane via different trafficking systems.
Microtubules are the largest cytoskeleton component, and they play a crucial role in a variety of cellular functions, including intracellular transport, organelle positioning, cell shape and motility, and centrosome and cilium formation Janke and Bulinski, To accomplish such diverse functions, microtubules associate with motor and non-motor proteins and undergo posttranslational modifications such as acetylation and detyrosination Janke and Bulinski, M2 ubiquitination plays an important role in influenza virus replication through facilitating the packaging of viral genome into virus particles and coordinating the timing of virus-induced host cell apoptosis and autophagy Su et al.
Moreover, ubiquitination-deficient mutant M2 K78R, had lower infectivity because it produced mainly defective virion particles that either lacked vRNPs or contained smaller amounts of internal viral components Su et al.
Influenza A viruses assembly requires the coordinated localization of different viral components at virus budding sites. M1 can interact with vRNPs, thus playing a key role in influenza virus assembly Schmitt and Lamb, Viruses carrying a SUMO-deficient M1 produce fewer infectious particles and form virions with more elongated or filamentous structures, indicating that a lack of M1 SUMOylation impairs viral morphogenesis Wu et al.
Palmitoylation of HA at the conserved carboxy-terminal of the cytoplasmic tail is required for efficient M1 recruitment and correlates with the ability to form infectious influenza virus particles Zurcher et al.
Acetylation is another PTM known to regulate protein function. Enhanced acetylation of microtubules increases IAV release from infected cells Husain and Harrod, Furthermore, the growth of NP acetylation-deficient KR mutant was severely attenuated in various cell types due to impaired virion release Giese et al.
Innate immunity acts as the first-line defense of host cells in restricting IAV replication Iwasaki and Pillai, During virus infection, the pattern recognition receptors PRR recognize pathogen-associated molecular patterns PAMPs in the invading viruses, leading to the activation of innate immune signaling cascades and the subsequent production of proinflammatory cytokines Iwasaki and Pillai, PTMs are involve in regulating innate immune signaling, and many viruses have evolved various mechanisms to usurp PTMs to counteract this host defense response Calistri et al.
For influenza virus, the activities of the immune-regulatory proteins, including NS1 Nogales et al. Production of type I IFNs, a family of anti-viral cytokines, is an integral component of the host innate immunity defense system, which play a critical role in inhibiting the accomplishment of the virus life cycle and impeding virus dissemination in vivo Garcia-Sastre et al. In contrast, NS1 from avian, human, and mouse-adapted influenza viruses inhibits RIG-I signaling in mouse cells only through binding to and blocking mouse Riplet Figure 3B Rajsbaum et al.
These findings for NS1 partly explain the host adaptation ability of influenza virus. Figure 3. B NS1 proteins from avian, human, and mouse-adapted influenza viruses can inhibit RIG-I signaling in mouse cells only through binding to and blocking mouse Riplet.
Moreover, as the infectious cycle progresses, dynamic phosphorylation of NS1 at T49 may alter NS1 activity Hsiang et al. ISGylation also limits influenza virus virulence. Figure 4. Mechanistically, through the endonuclease activity of its N terminus, PA interacts with IRF3 to suppress its phosphorylation, dimerization, and subsequent nuclear translocation and activation Yi et al.
This small protein acts to decrease the virulence of various influenza viruses through modulating the host innate immune response mainly through its host shutoff activity Hu et al. Mechanistically, the NatB-mediated N-terminal acetylation of PA-X contributes to this host shutoff activity and viral polymerase activity Oishi et al.
PB1-F2, another accessory protein, is a amino acid protein that is encoded as an internal open reading frame on the PB1 gene of some influenza viruses.
It contributes to the pathogenesis and comorbidity of influenza viruses mainly through manipulating apoptosis and innate immune responses, and enhancing the secondary bacterial infection Cheung et al.
In addition to modification of influenza virus proteins, modification of host proteins also acts as a major mechanism of the host cellular response to control influenza virus infection. IFITM3 is a host antiviral restriction factor that limits cellular infection with multiple notable viral pathogens and is especially crucial for the innate immune response against influenza virus Yount et al. S-palmitoylation controls IFITM3 clustering in membranes and contributes to its full antiviral activity against influenza virus Yount et al.
Therefore, the inhibition of these two negative regulatory modifications is speculated to increase antiviral activity Yount et al. TRIM28 has recently been implicated in contributing to inflammatory cytokine production during influenza virus infection Domingues et al.
Mitochondrial antiviral-signaling protein, a key mediator of IFN signaling, plays a critical role in host innate immunity against RNA viruses. Functional analyses revealed that these two PB1-F2 phosphorylation sites play an important role in influenza virus replication and promote cellular apoptosis in primary human monocytes Mitzner et al.
Moreover, the H7N9 influenza virus NS1 upregulated p53 expression through enhancing the phosphorylation levels of p53 and facilitating mitochondrial dysfunction, which may initiate NS1-induced apoptosis in human A cells Yan et al.
During the process of influenza virus host adaptation to different species, the viruses develop different immune evasion strategies in different species. To replicate efficiently in humans, avian influenza viruses must adapt their viral RNA polymerase vPol to human cells. Polymerase subunit PB2 is an important viral factor involved in influenza virus host adaption, and the EK mutation in this subunit is an adaptive marker of avian influenza virus in mammalian hosts.
However, it was proposed that to replicate efficiently in human cells, PB2 K vPol may increase the utilization efficiency of huANP32A rather than enhancing the binding activity of this protein Domingues and Hale, As an obligatory intracellular parasite, influenza virus heavily depends on host cellular factors and host cellular machineries to accomplish its life cycle and establish a successful infection.
PTMs allow for the dynamic and reversible control of protein functions by modulating the protein abundance, interactors, catalysis, or localization. PTMs also act in many signaling pathways, such as those related to gene regulation, epigenetics, differentiation, protein degradation, tumorigenesis, and immune signaling. Furthermore, PTMs are important parts of the host immune response and play essential roles in replication for many viruses.
For influenza virus, PTMs are involved in all steps of the viral replication cycle. The extensive usurpation of various host PTM-mediated pathways by influenza viruses at different stages of their life cycle emphasizes the crucial importance of these cellular machineries in cell physiology and function, and as a consequence, their importance for viral replication and viral diversion of cellular immunity.
Currently, manipulation of the host PTMs systems is emerging as a key theme in terms of viral pathogenesis. Accumulating research has found clear evidence of viral proteins that mimic or redirect host PTMs to modify the cellular environment and influence the balance between normal and pathogenic cellular signaling in favor of virus persistence or efficient replication.
However, given the wide sequence variation of the known candidates, it is a rather complicated task to systematically identify their viral analogs for ubiquitin or SUMO enzymes. In addition, proteins within released virions also contain multiple types of PTMs, although the function of these modifications within the virions is not currently well understood Hutchinson et al.
The complexity of the interplay between viruses and the PTMs systems poses an exciting challenge for future research aiming to unravel the crosstalk between all of these modifications and assess the outcomes at a global level, which may shed further light on the complexity of influenza virus biology.
Moreover, questions regarding whether all PTMs impact protein functions during virus infection or lead to dynamic changes in virus infection remain to be answered. There are also likely to be additional, as yet undiscovered, modes of post-translational regulation that occur on influenza virus proteins; their discovery will continue to add to our understanding of the role of PTMs systems during influenza virus infection.
Additionally, the mechanisms of how viruses hijack the host PTMs machinery in the process of their coevolution with the host cells remain unknown. Moreover, considering the broad-ranging importance of PTMs in regulating viral replication and the host innate immune response to influenza viruses, more systematic research on this area should bring us closer to controlling and using PTMs for the development of preventative or antiviral therapeutic strategies and may also facilitate the study of innate immunity.
Possible directions for future work could include clarifying the functional relevance of all PTMs involved in the specific stages of viral replication and systematically identifying the viral factors that interfere with PTMs. In addition, given the growing appreciation for various PTMs and their biological functions during microorganism infection, the development of new approaches for studying PTMs is necessary. Presently, mass spectrometry can concurrently identify different types of PTMs, including phosphorylation, acetylation, methylation, and ubiquitination.
Future work uncovering the dynamics of PTMs during microorganism infection will provide insight into the regulatory mechanisms and signaling pathways at a system-wide level. Top-down proteomics combined with time-resolved proteomics may reveal coincident modifications on proteins. Moreover, physiological approaches, such as the incorporation of unnatural amino acids with bio-orthogonal reactivity Neumann-Staubitz and Neumann, , analog-sensitive AS kinases technology Lopez et al.
JH and XL drafted and revised the manuscript. LZ contributed to the reference collection and analysis. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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We expect an increase of these approaches in the near future facilitated by improvement of data-analysis software, and bringing closer untargeted and targeted proteomics. However, the major strengths: absolute quantitation and high throughput possibilities must be kept to allow the move from relative to absolute quantification. BA conceived the idea, wrote the initial manuscript and made the figure, MW revised the manuscript and contributed with references, BU performed major revisions and contributed with valuable discussions.
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