Nucleophosmin: A Nucleolar Phosphoprotein Orchestrating Cellular Stress Responses

Nucleophosmin (NPM1) is a key nucleolar protein released from the nucleolus in response to stress stimuli. NPM1 functions as a stress regulator with nucleic acid and protein chaperone activities, rapidly shuttling between the nucleus and cytoplasm. NPM1 is ubiquitously expressed in tissues and can be found in the nucleolus, nucleoplasm, cytoplasm, and extracellular environment. It plays a central role in various biological processes such as ribosome biogenesis, cell cycle regulation, cell proliferation, DNA damage repair, and apoptosis. In addition, it is highly expressed in cancer cells and solid tumors, and its mutation is a major cause of acute myeloid leukemia (AML). This review focuses on NPM1’s structural features, functional diversity, subcellular distribution, and role in stress modulation.

NPM1 is associated with various pathological conditions.It is frequently overexpressed, altered, rearranged, and sporadically deleted in human cancers [46].Thus, there is little doubt that NPM1 is involved in human tumorigenesis, where its expression and gene integrity are frequently altered.NPM1 belongs to a novel gene category that functions as oncogenes and tumor suppressors.Depending on the expression level and gene dosage, either the partial functional loss of NPM1 or its aberrant overexpression can lead to neoplastic transformation through different mechanisms.Mutations in NPM1 occur in approximately one-third of patients with acute myeloid leukemia [47,48] and are clinically associated with leukocytosis, a high percentage of blasts, and extramedullary involvement [49].In addition, NPM1 has shown increased interest in radiotherapy, where its knockdown significantly reduces tumor cell survival after irradiation.Irradiation has been shown to induce the dephosphorylation of NPM1 at T199, T234, and T237, and its intracellular distribution between the nucleoli, nucleoplasm, and cytoplasm [50].Several studies have shown that the exposure of fibroblasts and lymphoblastoid cells to UV or γ-IR leads to the increased expression of NPM1 as an immediate early gene response, which is also induced by chromosomal instability [51,52].Thus, NPM1 appears to be a key determinant of nuclear homeostasis in protecting cells from radiation-induced apoptosis, but the elucidation of the underlying molecular mechanisms awaits future investigation.

Structure and Functions of NPM1
NPM1 is the most abundant isoform with 294 amino acids (aa) and an approximate molecular weight of 37 kDa [22,47].It consists of an N-terminal oligomerization domain (OD), a central histone binding domain (HBD), and a C-terminal nucleic acid binding domain (NBD) (Figure 1).hypoxia [39], heat shock [40], oxidative stress [41], UV irradiation [42], chemotherapeutic agents [43], and gamma irradiation [44,45].NPM1 is associated with various pathological conditions.It is frequently overexpressed, altered, rearranged, and sporadically deleted in human cancers [46].Thus, there is little doubt that NPM1 is involved in human tumorigenesis, where its expression and gene integrity are frequently altered.NPM1 belongs to a novel gene category that functions as oncogenes and tumor suppressors.Depending on the expression level and gene dosage, either the partial functional loss of NPM1 or its aberrant overexpression can lead to neoplastic transformation through different mechanisms.Mutations in NPM1 occur in approximately one-third of patients with acute myeloid leukemia [47,48] and are clinically associated with leukocytosis, a high percentage of blasts, and extramedullary involvement [49].In addition, NPM1 has shown increased interest in radiotherapy, where its knockdown significantly reduces tumor cell survival after irradiation.Irradiation has been shown to induce the dephosphorylation of NPM1 at T199, T234, and T237, and its intracellular distribution between the nucleoli, nucleoplasm, and cytoplasm [50].Several studies have shown that the exposure of fibroblasts and lymphoblastoid cells to UV or γ-IR leads to the increased expression of NPM1 as an immediate early gene response, which is also induced by chromosomal instability [51,52].Thus, NPM1 appears to be a key determinant of nuclear homeostasis in protecting cells from radiation-induced apoptosis, but the elucidation of the underlying molecular mechanisms awaits future investigation.

Structure and Functions of NPM1
NPM1 is the most abundant isoform with 294 amino acids (aa) and an approximate molecular weight of 37 kDa [22,47].It consists of an N-terminal oligomerization domain (OD), a central histone binding domain (HBD), and a C-terminal nucleic acid binding domain (NBD) (Figure 1).containing two nuclear export signals (NES1 and NES2), the central histone binding domain (HBD), comprising two acidic stretches A1 and A2 in addition to two nuclear localization signals (NLS1), and the C-terminal nucleic acid binding domain (NBD) containing the nuclear localization signal (NLS2) and the nucleolar localization signal (NoLS).Post-translational modifications (PTM) and related functions in addition to interacting partners, such as phosphorylation (green), acetylation (red), ubiquitination (dark blue), SUMOylation (light blue), and PARylation (magenta).For more details, see text.
The OD is highly conserved, methionine-rich, and contains a hydrophobic core [53] that organizes its oligomeric state.Thus, NPM1 exists in monomeric, pentameric, and decameric states [54].The pentameric NPM1 is normally localized to the nucleolus and is involved in nucleosome formation and chromatin remodeling [11,55,56].The N-terminal OD contains two canonical, leucine-rich nuclear export signals (NES; Figure 1) [16].NES1 regulates ribosomal subunit binding, protein synthesis, and cell proliferation.NES2 binds the RAN complex with exportin 1 (XPO1; also called CRM1) and regulates NPM1 ′ s association with centrosomes [16].The mutation of a potential phosphorylation site at threonine 95 in NES2 abolishes the NPM1 association and inhibition of centrosome duplication [57].The central unstructured, negatively charged HBD (Figure 1) mimics DNA structure for efficient binding to histones, preferentially to histone H3, to mediate nucleosome formation and chromatin decondensation [12,19,58,59].The central HBD contains one of the two nuclear localization signals (NLS) that act as importin α/β recognition sites and mediate nuclear localization of NPM1.The C-terminal positively charged NBD preferentially binds RNA.It can also bind double-stranded DNA [60,61] and ATP [62].In addition to an NLS, the NBD contains aromatic residues representing nucleolar localization signals (NoLS).Ribonucleolytic activity has been demonstrated at the 28S rRNA level in a common fraction of HBDs and NBDs [61,63], suggesting that NPM1 may play a role in ribosome maturation in addition to ribosome export from nucleoli.

NPM1 in Nucleolus
The nucleolus is a highly dynamic subnuclear compartment that undergoes major structural and compositional changes in response to growth signals, cellular status, and stress [3,4].Various factors, including DNA damage, nutrient deprivation, viral infection, and exposure to certain chemicals, can induce nucleolar stress.The nucleolus is considered a stress-sensing organelle in cells, and genotoxic stress induces nucleolar dynamic and functional changes.
NPM1 has been implicated in maintaining nucleolar structure by modulating liquidliquid phase separation (LLPS) through mechanisms driven by electrostatic interactions between either the negative and positive domains on NPM1 (homotypic) or between the positively and negatively charged domain of NPM1 on one side with the positively charged R motifs on other nucleolar proteins or negatively charged rRNA (heterotypic) [11].The interaction of NPM1 with SURF6 (Surfeit locus protein 6) is thought to modulate heterotypic vs. homotypic interactions [11,[103][104][105].Another nucleolar function of NPM1 is the initiation of the nucleolar stress response, where the nucleolus responds very early to stress and/or damage [2]; this early response occurs upon the modification of the nucleolar protein NPM1 by oxidation or S-glutathionylation (at cysteine residue 275), followed by the release and dissociation of the nucleolar protein NPM1 from the nucleolus and translocation to the nucleoplasm [48,106,107].

NPM1 in Nucleus
In the nucleus, NPM1 regulates cell survival through its interaction with PKB/AKT in response to growth factor stimulation and may begin to regulate and balance cell survival and apoptosis [108].On the other hand, the inhibition of apoptosis is achieved by binding to both nuclear PI(3,4,5)P3 and nuclear AKT, a complex that directly interacts with caspase-activated DNase and inhibits its DNA fragmentation activity [32,33] This NPM1-

NPM1 in Nucleus
In the nucleus, NPM1 regulates cell survival through its interaction with PKB/AKT in response to growth factor stimulation and may begin to regulate and balance cell survival and apoptosis [108].On the other hand, the inhibition of apoptosis is achieved by binding to both nuclear PI(3,4,5)P 3 and nuclear AKT, a complex that directly interacts with caspase-activated DNase and inhibits its DNA fragmentation activity [32,33] This NPM1-dependent process appears to be controlled by nuclear PI3K and its upstream regulator PIKE (PI3K enhancer), which is a nuclear GTPase [32].
NPM1 contains two NES signals and its shuttling properties are regulated via its interaction with RAN-CRM1, which are mainly involved in the regulation of centrosome duplication and spindle assembly by cyclin-CDK complexes, p53, BRCA1, and BRCA2 (Figure 2) [46,57,[109][110][111][112][113][114].NPM1 acts as a regulator of cell cycle progression; its overexpression induces the rapid entry of hematopoietic stem cells into the cell cycle by inhibiting the expression of many negative cell cycle regulators during the G1-S phase, while NPM1 depletion upregulates the expression of these negative regulators and induces cell cycle arrest during stress [115].The absence of NPM1 induces ploidy or uncontrolled centrosome amplification [114].NPM1 is recruited to the centrosome through its interaction with BRCA2 and ROCK2 [116,117].
Interestingly, the phosphorylation of NPM1 is critical for regulating its nuclear functions.The phosphorylation of NPM1 on serine 4 by PLK1 and NEK2A regulates its reassociation with the centrosome during mitosis [118].This process is also regulated by the NES of NPM1 and its interaction with RAN/CRM1, and the inhibition of its phosphorylation at T95, or mutation of the NES motif of NPM1, induces ploidy [6].In addition, the phosphorylation of T 199 with E/CDK2 is required for centrosome duplication and the regulation of G2/M cell cycle arrest through its interaction with p53 and the regulation of its interaction with GADD45 [47].Various stressors, such as UV, induce the rapid translocation of NPM1 to the nucleoplasm and its interaction with p53 and HDM2 [119].NPM1 regulates the cell cycle by transporting hyperphosphorylated Rb to the nucleolus, allowing for the dissociation between Rb and E2F protein, where E2F allows the cell cycle to proceed into the S phase [120].
On the other hand, NPM1 regulates and maintains genomic stability by regulating many repair mechanisms of the DNA damage response.It also regulates centrosome replication [6,97].NPM1 modulates base excision repair by forming a complex with the base excision repair proteins (APE1, FEN1, Polβ, and LIGL) [121,122], and the absence of NPM1 induces mislocalization between APE1, FEN1, and LIGL [12,97].NPM1 affects the stability and localization of many base excision repair proteins, such as the alternative reading frame tumor suppressor p14ARF and the apurinic/apyrimidinic endonuclease 1 (APE1) [97].Recently, it was shown that interaction between p14ARF and sirtuin7(SIRT7) blocks its interaction with NPM1 and subsequently induces its degradation via proteasomal activity [123].Another way in which genome stability is regulated is through the regulation of DNA double-strand breaks by interacting with both p53 and its negative regulator, the E3 ubiquitin ligase HDM2.This interaction protects p53 from degradation and prolongs its halflife during stress [119].NPM1 directly interacts with aa 639-1000 in BRCA2 and supports the double-strand break repair mechanism by forming BRCA2/RAD51 foci [116,124] Interestingly, the tions.The phosphory sociation with the cen NES of NPM1 and its tion at T95, or mutati phosphorylation of T regulation of G2/M ce its interaction with GA cation of NPM1 to the regulates the cell cycl ing for the dissociatio proceed into the S pha On the other han many repair mechanis lication [6,97].NPM1 excision repair protein induces mislocalizatio and localization of m frame tumor suppres [97].Recently, it was s its interaction with N tivity [123].Another w of DNA double-strand E3 ubiquitin ligase H its half-life during str supports the double- [116,124].It interferes main of Pol ղ and pre modification of NPM1 has been shown to tra the inhibition of this c Phosphorylated N does not affect the fo Rad51 and γ H2AX fo which are characteri [126,128,129].The dep tein phosphatase PP1 in response to UV irra pair mechanisms [48] volvement in HR and and preventing its proteasomal degradation [12,125].The post-translational modification of NPM1 regulates its DNA repair functions, where phosphorylated NPM1 has been shown to translocate to DNA double-strand breaks with γ-H2AX foci [126], and the inhibition of this colocalization sensitizes cells to ionizing radiation [127].
Phosphorylated NPM1 recruits to double-strand break Rad51 foci, and its depletion does not affect the formation of the foci but leads to their persistence.The analysis of Rad51 and γ H2AX foci in NPM1-null mouse embryonic fibroblasts has been conducted, which are characterized by persistent DNA damage without affecting cell survival [126,128,129].The dephosphorylation of NPM1 at threonines 199, 234, and 237 by the protein phosphatase PP1β due to the release of the retinoblastoma tumor suppressor (pRB) in response to UV irradiation is important for the activation of E2F1-dependent DNA repair mechanisms [48].NPM1 SUMOylation at k263 by p14ARF is required for NPM1 involvement in HR and RAD51 foci formation [79,124].

NPM1 in Cytoplasm
Although NPM1 should shuttle between the nucleus and cytoplasm, its predominant presence in the cytoplasm indicates abnormalities or severe mutations, including cancer (acute myeloid leukemia, AML) and viral infection [24].NPM1 in AML has insertional mutations that result in a frameshift that affects tryptophan residues on the nucleolar localization signal and converts it to a nuclear export signal, resulting in the aberrant cytoplasmic localization of NPM1 [16,48,130].The latter can be inhibited by CRM1 inhibitors [131,132].
NPM1 may also mediate actin cytoskeletal dynamics through the Ras-dependent hyperactivation of the mammalian target of the rapamycin (mTOR) protein [133].One of the most interesting functions of NPM1 is its role in regulating apoptosis.During stress, NPM1 suppresses the p53 apoptotic pathway by blocking the localization of p53 to mitochondria and preventing its translocation from the nucleus to mitochondria [37].Another anti-apoptotic activity of NPM1 appears through reducing the proteolytic activity of several caspases (3,6,8), where a remarkable decrease in caspase 8 activity has been reported in cultured cardiomyocytes (Figure 2) [12,134].On the other hand, NPM1 may regulate the pro-apoptotic activity in both intrinsic and extrinsic apoptosis.Its pro-apoptotic role in intrinsic apoptosis appears by its binding to activated BAX and the translocation of BAX to mitochondria.In this study, pro-apoptotic activity was monitored by detecting increased levels of mitochondrial cytochrome c release and caspase cascade activation with NPM1 downregulation using RNAi, and this pro-apoptotic effect is supported by the NFκB pathway and the direct interaction between NPM1 and other nucleolar proteins (RELA) [34,135,136].Nevertheless, the oncogenic mutant of NPM1 impairs mitochondrial function [137].Using pharmacological inhibitors of NPM1 to induce apoptosis in cancer cells, reports have monitored the anti-apoptotic properties of NPM1.The interaction of GAGE with NPM1 increases the stability of NPM1 and its resistance to interferon gamma-induced apoptosis [12,138].In extrinsic apoptosis, NPM1 showed anti-apoptotic activity through its fusion with retinoic acid receptor alpha (RARA) and by blocking the TNF-mediated activation of caspases 3 and 8 (CASP3/8) through its interaction with the tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD) (Figure 2) [139,140].
Interestingly, the disruption of the oligomerization state of NPM1, by disrupting its pentameric state formation, induces apoptosis and affects its subcellular localization [134].The inhibition of NPM1 nucleocytoplasmic shuttling also induces apoptosis [46,141,142].All these data together suggest a bipartite role of NPM1 in apoptosis or as having an antior pro-apoptotic effect like some apoptotic marker proteins (e.g., BCL2) [143].
The N-terminal domain of NPM1 is structurally responsible for the binding of many viral proteins, such as HIV Tat and Rev proteins, herpes simplex virus US11 [20,22], hepatitis B core proteins [144], and adenovirus basic core proteins (Figure 2) [145].As a result, NPM1 is involved in various stages of the viral life cycle, including the nuclear import of viral proteins and final assembly, making it a target for the treatment of various viral infections [11,23].

NPM1 at the Plasma Membrane and Its Secretion
The nucleolar proteins NPM1 and nucleolin have been reported at the inner leaflet of the plasma membrane using electron microscopy; these proteins have also been reported to interact with KRAS [146].Another interesting point is that at the plasma membrane, NPM1 binds and stabilizes activated KRAS nanoclusters to modulate signaling through the MAPK pathway [35].Furthermore, NPM1 regulates various cell signaling pathways such as proliferation and differentiation and cell survival via the MAPK pathway through its interaction with RAS proteins, specifically KRAS rather than HRAS.The acidic domain of NPM1 interacts with the basic domain of KRAS [35,146].
Several reports have drawn attention to the potential role of NPM1 in innate immunity.NPM1 was passively released into the extracellular milieu by necrotic or damaged cells but secreted by macrophages and monocytes [147].This secretion can be accompanied by miRNAs, and NPM1 is responsible for the stabilization of the secreted miRNAs [148].In addition, extracellular NPM1 acts as an inflammatory stimulator by inducing the production of inflammatory cytokines such as tumor necrosis factor (TNFα), IL-6, and IL-8 via ERK1/2 activation [149].TLR4 is a receptor that mediates NPM1 signaling, which requires NPM1 binding to myeloid differentiation protein-2 (MD-2).Thus, NPM1 activity may be useful in the treatment of TLR4-related diseases [147].

Conclusions
NPM1 is an essential multifunctional nucleolar protein.It diffuses from the nucleolus during stress to interfere with many stress-related processes such as genomic stability, cell cycle, and apoptosis.Its aberrant cytoplasmic localization is a sign of cancer (acute myeloid leukemia).Recently, NPM1 has been identified as a key protein in either pro/antiapoptotic activities and is responsible for the stabilization of many other proteins including tumor suppressor proteins; these findings developed our understanding of NPM1 beyond its implications in cancer prognosis and it has been identified as a primary therapeutic target.Approaches such as gene therapy to correct its mutations or targeting its interacting networks with small molecule inhibitors are being actively explored with a focus on cancer malignancy and cell response to radiotherapy.However, the molecular mechanisms underlying NPM1-related pathology are still poorly understood.Conversely, its anti-stress properties offer promising avenues for intervention in neurodegenerative diseases such as Huntington's disease by managing pathological aggregates, as well as many other stress-related diseases.
Advances in the understanding of the mechanisms governing NPM1 secretion, its interaction with different extracellular receptors, its involvement in external apoptosis and its potential fusion with other proteins will open new avenues.These developments could potentially transform NPM1 from a therapeutic target into a bioengineered recombinant drug candidate for various diseases.In addition, considering its chaperoning properties and its ability to modulate different stages of viral cellular processing, all of this together suggests that NPM1 is a key regulator of innate immunity and may serve as an immunotherapeutic or pro-inflammatory agent in many diseases as well as viral infections.

Figure 1 .Figure 1 .
Figure 1.Functional domains, post-translational modifications, and associated functions of the NPM family.NPM1 is composed of three functional domains, the N-terminal oligomerization domain (OD), containing two nuclear export signals (NES1 and NES2), the central histone binding domain (HBD), comprising two acidic stretches A1 and A2 in addition to two nuclear localization signals (NLS1), and the C-terminal nucleic acid binding domain (NBD) containing the nuclear Figure 1.Functional domains, post-translational modifications, and associated functions of the NPM family.NPM1 is composed of three functional domains, the N-terminal oligomerization domain (OD),

Figure 2 .
Figure 2. Schematic representation of the subcellular localization, associated functions, and interaction networks of NPM1.The nucleolus (fibrillar center (FC), dense fibril compartment (DFC), and granular compartment (GC)), nucleus(nucleoplasm), cytoplasm (cytosol), and outer layer represent the plasma membrane and extracellular milieu.For more details, see text.

Figure 2 .
Figure 2. Schematic representation of the subcellular localization, associated functions, and interaction networks of NPM1.The nucleolus (fibrillar center (FC), dense fibril compartment (DFC), and granular compartment (GC)), nucleus(nucleoplasm), cytoplasm (cytosol), and outer layer represent the plasma membrane and extracellular milieu.For more details, see text.
. It interferes in translesion DNA synthesis by interacting with the catalytic domain of Pol