Highly Similar Tetramerization Domains from the p53 Protein of Different Mammalian Species Possess Varying Biophysical, Functional and Structural Properties.

The p53 protein is a transcriptional regulatory factor and many of its functions require that it forms a tetrameric structure. Although the tetramerization domain of mammalian p53 proteins (p53TD) share significant sequence similarities, it was recently shown that the tree shrew p53TD is considerably more thermostable than the human p53TD. To determine whether other mammalian species display differences in this domain, we used biophysical, functional, and structural studies to compare the properties of the p53TDs from six mammalian model organisms (human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum). The results indicate that the p53TD from the opossum and tree shrew are significantly more stable than the human p53TD, and there is a correlation between the thermostability of the p53TDs and their ability to activate transcription. Structural analysis of the tree shrew and opossum p53TDs indicated that amino acid substitutions within two distinct regions of their p53TDs can dramatically alter hydrophobic packing of the tetramer, and in particular substitutions at positions corresponding to F341 and Q354 of the human p53TD. Together, the results suggest that subtle changes in the sequence of the p53TD can dramatically alter the stability, and potentially lead to important changes in the functional activity, of the p53 protein.

Biomineralization through a Symmetry-Controlled Oligomeric Peptide.

Biomineralization peptides are versatile tools for generating nanostructures since they can make specific interactions with various inorganic metals, which can lead to the formation of intricate nanostructures. Previously, we examined the influence that multivalency has on inorganic structures formed by p53 tetramer-based biomineralization peptides and noted a connection between the geometry of the peptide and its ability to regulate nanostructure formation. To investigate the role of multivalency in nanostructure formation by biomineralization peptides more thoroughly, silver biomineralization peptides were engineered by linking them to additional self-assembling molecules based on coiled-coil peptides and multistranded DNA oligomers. Under mild reducing conditions at room temperature, these engineered biomineralization peptides self-assembled and formed silver nanostructures. The trimeric forms of the biomineralization peptides were the most efficient in forming a hexagonal disk nanostructure, with both the coiled-coil peptide and DNA-based multimeric forms. Together, the results suggest that the spatial arrangement of biomineralization peptides plays a more important role in regulating nanostructure formation than their valency.

Bilayer Hydrogel Composed of Elastin-Mimetic Polypeptides as a Bio-Actuator with Bidirectional and Reversible Bending Behaviors.

Biologically derived hydrogels have attracted attention as promising polymers for use in biomedical applications because of their high biocompatibility, biodegradability, and low toxicity. Elastin-mimetic polypeptides (EMPs), which contain a repeated amino acid sequence derived from the hydrophobic domain of tropoelastin, exhibit reversible phase transition behavior, and thus, represent an interesting starting point for the development of biologically derived hydrogels. In this study, we succeeded in developing functional EMP-conjugated hydrogels that displayed temperature-responsive swelling/shrinking properties. The EMP-conjugated hydrogels were prepared through the polymerization of acrylated EMP with acrylamide. The EMP hydrogel swelled and shrank in response to temperature changes, and the swelling/shrinking capacity of the EMP hydrogels could be controlled by altering either the amount of EMP or the salt concentration in the buffer. The EMP hydrogels were able to select a uniform component of EMPs with a desired and specific repeat number of the EMP sequence, which could control the swelling/shrinking property of the EMP hydrogel. Moreover, we developed a smart hydrogel actuator based on EMP crosslinked hydrogels and non-crosslinked hydrogels that exhibited bidirectional curvature behavior in response to changes in temperature. These thermally responsive EMP hydrogels have potential use as bio-actuators for a number of biomedical applications.

Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells.

The tumor suppressor p53 (also known as TP53) plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function; these encode input information and are decoded to induce distinct cellular phenotypes. However, it remains unclear to what extent the temporal dynamics of p53 reflect the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single-cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 after etoposide treatment is highly dependent on the cell cycle but this is not seen after UV exposure. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway.

Ribosomal protein uL30 undergoes phase separation with nucleophosmin and regulates nucleolar formation in the absence of RNA.

The nucleolus is a membrane-less structure that exists in the nucleus of cells and plays a crucial role in ribosome biogenesis. It is known to be formed through liquid-liquid phase separation (LLPS) caused by the interaction of various nucleolar proteins and nucleic acids. Recently, many studies on LLPS with nucleolar proteins in the presence of RNA showed the importance of electrostatic interactions and cation-pi interactions among RNA and intrinsically disordered regions of proteins. However, it is reported that the initiation of nucleolar formation is RNA polymerase I-independent. The mechanism of nucleolar formation in the early stage remains obscure. In this study, we showed for the first time that the ribosomal protein uL30 and a major nucleolar protein, nucleophosmin (NPM) formed liquid droplets in vitro in the absence of RNA. The liquid droplet formation with uL30 and NPM may be derived from the interaction between the basic regions of uL30 and acidic regions of the oligomeric NPM. The knockdown of uL30 in cells significantly reduced the number of nucleoli, while it did not alter the protein level of NPM. The results showed that LLPS and nucleolar formation were affected by changes in uL30 levels. Our results suggest that the protein-protein interaction between nucleolar proteins may play an important role in nucleolar formation in the early stages when the rRNA content is very low.

Lipid Droplet Formation Is Regulated by Ser/Thr Phosphatase PPM1D via Dephosphorylation of Perilipin 1.

Hypertrophy and hyperplasia of white adipocytes induce obesity, leading to diseases such as type 2 diabetes and hypertension, and even cancer. Hypertrophy of white adipocytes is attributed to the excessive storage of the energy form of triglycerides in lipid droplets (LDs). LDs are fat storage organelles that maintain whole-body energy homeostasis. It is important to understand the mechanism of LD formation for the development of obesity therapy; however, the regulatory mechanisms of LD size and formation are not fully understood. In this study, we demonstrated that the PPM family phosphatase PPM1D regulates LD formation. PPM1D specific inhibitor, SL-176 significantly decreased LD formation via two different pathways: dependent of and independent of adipocyte-differentiation processes. In the mature white adipocytes after differentiation, LD formation was found to be controlled by PPM1D via dephosphorylation of Ser511 of perilipin 1. We found that inhibition of PPM1D in mature white adipocytes significantly reduced the size of the LDs via dephosphorylation of Ser511 of perilipin 1 but did not change the lipolysis sensitivity and the total amount of lipid in cells. Collectively, the results of this study provide evidence that PPM1D plays an important role in LD formation in mature adipocytes.

Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1.

SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO-SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.

PPM1D Is a Therapeutic Target in Childhood Neural Tumors.

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.

Role of active site arginine residues in substrate recognition by PPM1A.

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.

Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors.

Protein phosphatases and kinases control multiple cellular events including proliferation, differentiation, and stress responses through regulating reversible protein phosphorylation, the most important post-translational modification. Members of metal-dependent protein phosphatase (PPM) family, also known as PP2C phosphatases, are Ser/Thr phosphatases that bind manganese/magnesium ions (Mn2+/Mg2+) in their active center and function as single subunit enzymes. In mammals, there are 20 isoforms of PPM phosphatases: PPM1A, PPM1B, PPM1D, PPM1E, PPM1F, PPM1G, PPM1H, PPM1J, PPM1K, PPM1L, PPM1M, PPM1N, ILKAP, PDP1, PDP2, PHLPP1, PHLPP2, PP2D1, PPTC7, and TAB1, whereas there are only 8 in yeast. Phylogenetic analysis of the DNA sequences of vertebrate PPM isoforms revealed that they can be divided into 12 different classes: PPM1A/PPM1B/PPM1N, PPM1D, PPM1E/PPM1F, PPM1G, PPM1H/PPM1J/PPM1M, PPM1K, PPM1L, ILKAP, PDP1/PDP2, PP2D1/PHLPP1/PHLPP2, TAB1, and PPTC7. PPM-family members have a conserved catalytic core region, which contains the metal-chelating residues. The different isoforms also have isoform specific regions within their catalytic core domain and terminal domains, and these regions may be involved in substrate recognition and/or functional regulation of the phosphatases. The twenty mammalian PPM phosphatases are involved in regulating diverse cellular functions, such as cell cycle control, cell differentiation, immune responses, and cell metabolism. Mutation, overexpression, or deletion of the PPM phosphatase gene results in abnormal cellular responses, which lead to various human diseases. This review focuses on the structures and biological functions of the PPM-phosphatase family and their associated diseases. The development of specific inhibitors against the PPM phosphatase family as a therapeutic strategy will also be discussed.

Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins.

The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner.

The interactions between SUMO proteins and SUMO-interacting motif (SIM) in nuclear bodies formed by the promyelocytic leukemia (PML) protein (PML-NBs) have been shown to be modulated by either phosphorylation of the SIMs or acetylation of SUMO proteins. However, little is known about how this occurs at the atomic level. In this work, we examined the role that acetylation of SUMO1 plays on its binding to the phosphorylated SIMs (phosphoSIMs) of PML and Daxx. Our results demonstrate that SUMO1 binding to the phosphoSIM of either PML or Daxx is dramatically reduced by acetylation at either K39 or K46. However, acetylation at K37 only impacts binding to Daxx. Structures of acetylated SUMO1 variants bound to the phosphoSIMs of PML and Daxx demonstrate that there is structural plasticity in SUMO-SIM interactions. The plasticity observed in these structures provides a robust mechanism for regulating SUMO-SIM interactions in PML-NBs using signaling generated post-translational modifications.

The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein.

The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination of biophysical and modeling studies, we demonstrate that the tetramerization domain of the tree shrew p53 protein is considerably more stable than the corresponding domain from humans despite sharing almost 90% sequence identity. Based on modeling and mutagenesis studies, we determine that a glutamine to methionine substitution at position 354 plays a key role in this difference. Given the link between stability of the p53 tetramerization domain and its transcriptional activity, the results suggest that this enhanced stability could lead to important consequences at p53-regulated genes in the tree shrew.

Inhibition of lipid droplet formation by Ser/Thr protein phosphatase PPM1D inhibitor, SL-176.

Obesity is a worldwide public health problem, which is associated with various severe diseases including diabetes, hypertension, atherosclerosis, and cancer. Recent studies have revealed that combination treatment of several different compounds using low doses is effective to reduce side effects. Thus, there is a need to develop an efficient inhibitor for reducing lipid droplets with a divergent target/pathway. Ser/Thr protein phosphatase PPM1D is involved in cellular metabolic processes and is a promising target for anti-obesity treatment. We have previously developed a potent and specific PPM1D inhibitor, SL-176. In this study, we demonstrated that significant reduction of lipid droplet formation in adipocytes by the PPM1D specific inhibitor, SL-176. Using Oil-red O staining and fluorescent imaging of lipid droplet, we found that treatment of SL-176 significantly suppressed lipid droplet formation of 3T3-L1 cells both in amount and in size. SL-176 also repressed mRNA and protein expression of PPARγ and C/EBPα, adipogenic markers, at nontoxic conditions. Thus, SL-176 is a unique and potent inhibitor of lipid droplet formation that acts via PPM1D, a novel target toward inhibiting adipocyte differentiation.

Inhibition of protein phosphatase PPM1D enhances retinoic acid-induced differentiation in human embryonic carcinoma cell line.

The protein phosphatase PPM1D (Wip1) was originally identified as a p53 target product. Activation of PPM1D through various mechanism promotes the tumorigenic potential of various cancers by suppressing p53 and other DNA damage response proteins. New functions of PPM1D have recently been revealed in physiological processes such as cell differentiation. However, the regulatory mechanisms of signalling pathway to maintain stemness and induce cell differentiation are still unclear. Here we report that PPM1D modulates retinoic acid (RA) signalling. PPM1D knockdown resulted in decreased alkaline phosphatase activity of the human teratocarcinoma cell line NT2/D1. Inhibition of PPM1D-induced cell differentiation and decreased gene expression of the stem cell marker Oct-4 (POU5F1). RA-induced cell differentiation was promoted by reducing PPM1D activity. RA treatment elicited activation of the MEK-ERK pathway and induced rapid and transient activation of the extracellular signal-regulated kinase 1/2 (ERK-1/2). PPM1D dephosphorylated a phosphopeptide with the TEY motif in ERK-1/2 in vitro. Moreover, phosphorylation of ERK-1/2 was facilitated by PPM1D inhibition. Our study shows that PPM1D plays an important role in maintaining the undifferentiation state and a new function in RA-induced ERK regulation and cell differentiation.

Interferon stimulation creates chromatin marks and establishes transcriptional memory.

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

Mutant p53-Expressing Cells Undergo Necroptosis via Cell Competition with the Neighboring Normal Epithelial Cells.

p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo systems, we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer. When mutant p53 cells alone are present, cell death does not occur, indicating that necroptosis results from cell competition with the surrounding normal cells. Furthermore, when p53R273H mutation occurs within RasV12-transformed epithelia, cell death is strongly suppressed and most of the p53R273H-expressing cells remain intact. These results suggest that the order of oncogenic mutations in cancer development could be dictated by cell competition.

Synergic Strategies for the Enhanced Self-Assembly of Biomineralization Peptides for the Synthesis of Functional Nanomaterials.

INTRODUCTION: Peptide-mediated biomineralization is a promising bioinspired technique of nanostructure formation. The biomineralization peptide is responsible for the regulation of the growth and morphology of the inorganic nanostructure. The 3D properties of the biomineralization peptide is a crucial factor in which the success of creating functional nanomaterials depends on. However, given the relatively short sequence of most biomineralization peptides, controlling the multivalency and spatial orientation of the peptide can be a challenging endeavor. OBJECTIVE: This mini-review details recent advances in enhancing the self-assembly and 3D properties of the biomineralization peptide. The design and creation of fusion peptides is highlighted, which involves the conjugation of the biomineralization peptide with a control element. The control element is responsible for directing the self-assembly of the biomineralization peptide. CONCLUSION: A variety of control elements that can direct the self-assembly of biomineralization peptides are available. The control element can be a small organic molecule such as a biphenyl, or a peptide segment such as the p53 tetramerization domain or the amyloid peptide. The high diversity of existing control elements further increases the ability of peptide-mediated biomineralization to create functional nanomaterials.

Oligomerization enhances the binding affinity of a silver biomineralization peptide and catalyzes nanostructure formation.

Binding affinity and specificity are crucial factors that influence nanostructure control by biomineralization peptides. In this paper, we analysed the role that the oligomeric state of a silver biomineralization peptide plays in regulating the morphology of silver nanostructure formation. Oligomerization was achieved by conjugating the silver specific TBP biomineralization peptide to the p53 tetramerization domain peptide (p53Tet). Interestingly, the TBP-p53Tet tetrameric peptide acted as a growth catalyst, controlling silver crystal growth, which resulted in the formation of hexagonal silver nanoplates without consuming the peptide. The TBP-p53Tet peptide caps the surface of the silver crystals, which enhances crystal growth on specific faces and thereby regulates silver nanostructure formation in a catalytic fashion. The present findings not only provide an efficient strategy for controlling silver nanostructure formation by biomineralization peptides, but they also demonstrate that in this case the oligomeric peptides play a unique catalytic role.

Inhibition of Ser/Thr phosphatase PPM1D induces neutrophil differentiation in HL-60 cells.

Protein phosphatase Magnesium-dependent 1, Delta (PPM1D) is a wild-type p53-inducible Ser/Thr phosphatase that acts as a negative regulator of the p53 tumor suppressor. Gene amplification and overexpression of PPM1D have been reported in various cancers including leukemia and neuroblastoma. Therefore, PPM1D is a promising target in cancer therapy. It has been reported that PPM1D knockout mice exhibit neutrophilia in blood and show a defective immune response. Here, we found that inhibition of PPM1D induced neutrophil differentiation of human promyelocytic leukemia cell line HL-60. The combination of a PPM1D inhibitor and all-trans retinoic acid significantly increased their differentiation efficiency. The PPM1D inhibitor also induced G1 arrest in HL-60 cells. Our results suggest that PPM1D may be a potential therapeutic target for blood cell diseases including leukemia.