C-terminal Regulatory Domain Research Articles

Sequence Characteristics and Expression Analysis of GhCIPK23 Gene in Upland Cotton (Gossypium hirsutum L.).

CIPK (calcineurin B-like-interacting protein kinase) is a kind of serine/threonine protein kinase widely existing in plants, and it plays an important role in plant growth and development and stress response. To better understand the biological functions of the GhCIPK23 gene in upland cotton, the coding sequence (CDS) of the GhCIPK23 gene was cloned in upland cotton, and its protein sequence, evolutionary relationship, subcellular localization, expression pattern and cis-acting elements in the promoter region were analyzed. Our results showed that the full-length CDS of GhCIPK23 was 1368 bp, encoding a protein with 455 amino acids. The molecular weight and isoelectric point of this protein were 50.83 KDa and 8.94, respectively. The GhCIPK23 protein contained a conserved N-terminal protein kinase domain and C-terminal regulatory domain of the CIPK gene family member. Phylogenetic tree analysis demonstrated that GhCIPK23 had a close relationship with AtCIPK23, followed by OsCIPK23, and belonged to Group A with AtCIPK23 and OsCIPK23. The subcellular localization experiment indicated that GhCIPK23 was located in the plasma membrane. Tissue expression analysis showed that GhCIPK23 had the highest expression in petals, followed by sepals, and the lowest in fibers. Stress expression analysis showed that the expression of the GhCIPK23 gene was in response to drought, salt, low-temperature and exogenous abscisic acid (ABA) treatment, and had different expression patterns under different stress conditions. Further cis-acting elements analysis showed that the GhCIPK23 promoter region had cis-acting elements in response to abiotic stress, phytohormones and light. These results established a foundation for understanding the function of GhCIPK23 and breeding varieties with high-stress tolerance in cotton.

Emulsion-based evolution of Escherichia coli for higher growth yield on D-xylose identifies central role of cyclic AMP

D-xylose is an abundant sugar found in plant biomass and can be used as a renewable feedstock for the microbial production of diverse biofuels and bioproducts. However, D-xylose metabolism is slow in many industrial microorganisms, at least as compared to glucose metabolism. Not surprisingly, a number of approaches have been developed for improving D-xylose metabolism in diverse microorganisms. In this work, we applied a previously developed evolution strategy based on media-in-oil emulsions for improving the growth yield of Escherichia coli NCM3722 on D-xylose. After 30 rounds of evolutions, we isolated multiple mutants with increased growth yield on D-xylose. In addition, we also observed similar increases in the growth rate. Three mutants were selected for whole-genome sequencing. Two mutants had an amber stop mutation in adenylate cyclase, which truncates nearly 60% of the enzyme. However, the ability of this mutant to grow on xylose indicated that truncated enzyme, lacking the C-terminal regulatory domain, is still active. The other mutant had a point mutation in the cyclic AMP receptor protein (CRP), near the high affinity binding site for cyclic AMP. Both mutations, when introduced into wild type E. coli, were able to increase the growth yields at levels similar to the isolated mutants. In addition to D-xylose, these mutant strains and their genetic mimics also exhibited higher growth rates and yields on glucose, lactose, and L-arabinose. These results suggest that the improved growth rates and yields are due to changes in the production and sensing of intracellular cyclic AMP concentrations and also suggest native concentrations are suboptimal with respect to the growth rate and yield under the growth conditions tested. Collectively, these results may prove useful for engineering strains of E. coli for high-density fermentations or protein production.

AMPK promotes Arf6 activation in a kinase-independent manner upon glucose starvation.

ABSTRACTAMP-activated protein kinase (AMPK) is a crucial cellular nutrient and energy sensor that maintains energy homeostasis. AMPK also governs cancer cell invasion and migration by regulating gene expression and activating multiple cellular signaling pathways. ADP-ribosylation factor 6 (Arf6) can be activated via nucleotide exchange by guanine-nucleotide-exchange factors (GEFs), and its activation also regulates tumor invasion and migration. By studying GEF-mediated Arf6 activation, we have elucidated that AMPK functions as a noncanonical GEF for Arf6 in a kinase-independent manner. Moreover, by examining the physiological role of the AMPK–Arf6 axis, we have determined that AMPK activates Arf6 upon glucose starvation and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) treatment. We have further identified the binding motif in the C-terminal regulatory domain of AMPK that is responsible for promoting Arf6 activation and, thus, inducing cell migration and invasion. These findings reveal a noncanonical role of AMPK in which its C-terminal regulatory domain serves as a GEF for Arf6 during glucose deprivation.

The first identified invertebrate LGP2-like homolog gene in the Pacific oyster Crassostrea gigas

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster, Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN, AP-1, and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology.

Scallop RIG-I-like receptor 1 responses to polyinosinic:polycytidylic acid challenge and its interactions with the mitochondrial antiviral signaling protein

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a class of pattern recognition receptors located in the cytoplasm that play a key role in antiviral innate immunity in animals. However, few studies have been conducted on the function of RLR proteins in invertebrates. In this study, the complete coding sequence of the RLR gene of the Zhikong scallop, Chlamys farreri, was obtained and named CfRLR1 with an aim to study the response of CfRLR1 to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and the interaction between the CfRLR1 and C. farreri mitochondrial antiviral signaling (MAVS) protein. Sequence analysis revealed that CfRLR1 encodes 1161 amino acids, and the encoded protein covers two tandem caspase activation and recruitment domains (CARDs), a helicase domain, and a C-terminal regulatory domain. Phylogenetic analysis revealed that CfRLR1 belongs to the RLR family of mollusks. Quantitative real-time polymerase chain reaction showed that CfRLR1 mRNA was expressed in all tested tissues, with its highest expression observed in feet and gill tissues. Furthermore, CfRLR1 expression in the gill tissues was significantly induced after the poly (I:C) challenge. Finally, the results of co-immunoprecipitation and yeast two-hybrid assays revealed that CfRLR1 can bind to the CfMAVS protein via CARD–CARD interactions. Overall, our results elucidate the immune function of invertebrate RLR proteins and provide valuable information on viral disease control for scallop farming.

Interferon Regulatory Factors Functioned as Activators of the Interferon Pathway in the Scallop Chlamys farreri

Interferon regulatory factors (IRFs) are a family of transcription factors that control many facets during innate and adaptive immune responses. Vertebrate IRFs play important roles in regulating the expression of interferons (IFNs) and IFN-stimulated genes, while only limited studies were conducted on invertebrate IRFs. In the present study, four IRF family genes (CfIRF1, CfIRF1-like, CfIRF2, and CfIRF8) were identified from Zhikong scallop (Chlamys farreri) through whole-genome scanning. CfIRFs contain a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. CfIRFs were constitutively expressed during development as well as in adult tissues, especially in hepatopancreas, hemolymph, gill, and mantle. In hemolymph, qRT-PCR analysis revealed that CfIRF1, CfIRF1-like, and CfIRF2 were significantly upregulated in response to Vibrio anguillarum infection, and their encoding proteins could translocate into nucleus. Dual-luciferase reporter assay on CfIRF1, CfIRF1-like, and CfIRF2 showed that these three proteins were capable to induce a strong activation of ISRE promoters. Notably, in comparison with CfIRF1 and CfIRF1-like, CfIRF2 showed the most sensitive responses in coping with V. anguillarum, and consistently, CfIRF2 exhibited the most significant activation on ISRE. This study would provide valuable information for the innate immune roles of the IRF gene family in bivalve molluscs.

FOXO4 interacts with p53 TAD and CRD and inhibits its binding to DNA.

Transcription factor p53 protects cells against tumorigenesis when subjected to various cellular stresses. Under these conditions, p53 interacts with transcription factor Forkhead box O (FOXO) 4, thereby inducing cellular senescence by upregulating the transcription of senescence-associated protein p21. However, the structural details of this interaction remain unclear. Here, we characterize the interaction between p53 and FOXO4 by NMR, chemical cross-linking, and analytical ultracentrifugation. Our results reveal that the interaction between p53 TAD and the FOXO4 Forkhead domain is essential for the overall stability of the p53:FOXO4 complex. Furthermore, contacts involving the N-terminal segment of FOXO4, the C-terminal negative regulatory domain of p53 and the DNA-binding domains of both proteins stabilize the complex, whose formation blocks p53 binding to DNA but without affecting the DNA-binding properties of FOXO4. Therefore, our structural findings may help to understand the intertwined functions of p53 and FOXO4 in cellular homeostasis, longevity, and stress response.

When more is less: heritable gain-of-function chk1 mutations impair human fertility.

Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible.

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq.

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

The role of SnRK2 in the response to stress, the growth and development of plants

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) is a specific Ser/Thr protein kinase in plants. SnRK2 can regulate the expression of downstream genes or transcription factors through phosphorylation of substrates to achieve stress resistance regulation in different tissue parts, and make plants adapt to adverse environment. SnRK2 has a small number of members and a molecular weight of about 40 kDa, and contains a conserved N-terminal kinase domain and a divergent C-terminal regulatory domain, which plays an important role in the expression of enzyme. This review summarized the recent research progresses on the discovery, structure, and classification of SnRK2, and its function in response to various stresses and in regulating growth and development, followed by prospecting the future research direction of SnRK2. This review may provide a reference for genetic improvement of crop stress resistance.

Atomic structure of the regulatory TGS domain of Rel protein from Mycobacterium tuberculosis and its interaction with deacylated tRNA.

The stringent response is critical for the survival of Mycobacterium tuberculosis (Mtb) under nutrient starvation. The mechanism is mediated by a GTP pyrophosphokinase known as Rel, containing N-terminal synthetase and hydrolase domains and C-terminal regulatory domains, which include the TGS domain (ThrRS, GTPase, and SpoT proteins) that has been proposed to activate the synthetase domain via interaction with deacylated tRNA. Here, we present the NMR solution structure of the Mtb Rel TGS domain (MtRel TGS), consisting of five antiparallel β-strands and one helix-loop-helix motif. The interaction of MtRel TGS with deacylated tRNA is shown, indicating the critical amino acids of MtRel TGS in tRNA binding, and presenting the first structural evidence of MtRel TGS binding to deacylated tRNA in solution in the absence of the translational machinery.

ERAP1 binds peptide C-termini of different sequences and/or lengths by a common recognition mechanism

Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays a key role in controlling the immunopeptidomes available for presentation by MHC (major histocompatibility complex) molecules, thus influences immunodominance and cell-mediated immunity. It carries out this critical function by a unique molecular ruler mechanism that trims antigenic precursors in a peptide-length and sequence dependent manner. Acting as a molecular ruler, ERAP1 is capable of concurrently binding antigen peptide N- and C-termini by its N-terminal catalytic and C-terminal regulatory domains, respectively. As such ERAP1 can not only monitor substrate’s lengths, but also exhibit a degree of sequence specificity at substrates’ N- and C-termini. On the other hand, it also allows certain sequence and length flexibility in the middle part of peptide substrates that is critical for shaping MHC restricted immunopeptidomes. Here we report structural and biochemical studies to understand the molecular details on how ERAP1 can accommodate side chains of different anchoring residues at the substrate’s C-terminus. We also examine how ERAP1 can accommodate antigen peptide precursors with length flexibility. Based on two newly determined complex structures, we find that ERAP1 binds the C-termini of peptides similarly even with different substrate sequences and/or lengths, by utilizing the same hydrophobic specificity pocket to accommodate peptides with either a Phe or Leu as the C-terminal anchor residue. In addition, SPR (surface plasmon resonance) binding analyses in solution further confirm the biological significance of these peptide-ERAP1 interactions. Similar to the binding mode of MHC-I molecules, ERAP1 accommodates for antigenic peptide length difference by allowing the peptide middle part to kink or bulge at the middle of its substrate binding cleft. This explains how SNP coded variants located at the middle of ERAP1 substrate binding cleft would influence the antigen pool and an individual’s susceptibility to diseases.

Abstract 2160: Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression

Abstract Programmed Cell Death Protein 4 (PDCD4), a well-known tumor suppressor, acts to inhibit translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. EIF4A tends to target mRNAs with structured 5'-UTR. PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 may inhibit translation of certain mRNAs via directly binding. A previous study has demonstrated that PDCD4 inhibits translation of p53 mRNA and treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in down-regulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism in which p53 regulates expression of Pdcd4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. First, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased PDCD4 protein level. Secondly, p53 dose-dependently decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations on p53 (R273H: contact mutant, hotspot and R175H: conformational mutant, hotspot) abolished p53-mediated PDCD4 repression. Furthermore, using Tet-on expression system, PDCD4 protein level was decreased when WT p53, but not hotspot mutants of p53, was expressed in H1299 cells. Finally, mutations on DNA-binding domain, but not C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4 and the relationship between of p53 and PDCD4 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Andrew P. George, William H. Yang, Chiung-Min Wang, Richard H. Yang. Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2160.

Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

Understanding molecular mechanism of PLCe regulation by small G‐proteins

Cardiovascular disease is the leading cause of death in the United States. The phospholipase C (PLC) family of enzymes catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control cellular function and behavior. The PLCe subfamily is required for normal cardiovascular function, where it is regulated through direct interactions with the RhoA and Rap1A small GTPases, which are activated in response to G protein-coupled receptor (GPCR stimulation). RhoA activates PLCe at the plasma membrane, whereas Rap1A translocates and activates PLCe at the perinuclear membrane. However, the domains of PLCe involved in G protein binding and activation, and translocation to different subcellular membranes is largely unknown. In this work, we use cell-based activity assays, epifluorescence, and confocal microscopy to identify the domains of PLCe involved in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Our preliminary studies demonstrate that the unique N- and C-terminal regulatory domains of PLCe dictate its location within the cell, and contribute differently to basal and G protein-dependent activity. These studies will provide needed insights into the regulation and localization of PLCe in cells, which is critical for its roles in cardiovascular function.

Characterization of Cystathionine β-Synthase TtCbs1 and Cysteine Synthase TtCsa1 Involved in Cysteine Biosynthesis in Tetrahymena thermophila.

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β-synthase and cystathionine γ-lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5'-phosphate-dependent enzymes, cystathionine β-synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N-terminal heme binding domain, catalytic domain, and C-terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His-TtCbs1 and His-TtCsa1 were expressed and purified invitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high Km for O-acetylserine and low Km for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.

Zn2+ modulates in vitro phase separation of TDP-432C and mutant TDP-432C-A315T C-terminal fragments of TDP-43 protein implicated in ALS and FTLD-TDP diseases

TDP-43 proteinopathy is implicated in the neurodegenerative diseases, ALS and FTLD-TDP. Metal ion dyshomeostasis is observed in neurodegenerative diseases including ALS. Previously, mice expressing A315T familial ALS TDP-43 mutant showed elevated spinal cord Zn2+ levels. Recently, Zn2+ was observed to modulate the in vitro amyloid-like aggregation of the TDP-43's RRM12 domains. As a systematic knowledge of the TDP-43's interaction with Zn2+ is lacking, we in silico predicted potential Zn2+ binding sites in TDP-43 and estimated their relative solvent accessibilities. Zn2+ binding sites were predicted in the TDP-43's N-terminal domain, in the linker region between RRM1 and RRM2 domain, within RRM2 domain and at the junction of the RRM2 and C-terminal domain (CTD), but none in the 311–360 region of CTD. Furthermore, we found that Zn2+ promotes the in vitro thioflavin-T-positive aggregations of C-terminal fragments (CTFs) termed TDP-432C and TDP-432C-A315T that encompass the RRM2 and CTD domains. Also, while the Alexa-fluor fluorescently labelled TDP-432C and TDP-432C-A315T proteins manifested liquid-like spherical droplets, Zn2+ caused a solid-like phase separation that was not ameliorated even by carboxymethylation of the free cysteines thereby implicating the other Zn2+-binding residues. The observed Zn2+-promoted TDP-43 CTF's solid-like phase separation can be relevant to the Zn2+ dyshomeostasis in ALS and FTLD-TDP.

Regulation of catalytic activity and nucleolar localization of rat DNA topoisomerase IIα through its C-terminal domain.

Type II DNA topoisomerase (topo II) catalyzes double-stranded DNA cleavage and re-ligation, thus solving problems in DNA topology. Vertebrates have two isozymes (α and β). Recently, the C-terminal regulatory domain (CRD), which regulates catalytic activity and subnuclear localization by associating with RNA, was identified within the C-terminal domain (CTD) of rat topo IIβ. In contrast, it is unclear whether a β CRD-like domain is present in the CTD of topo IIα. In this study, we aimed to identify an RNA-mediated regulatory domain in the rat topo IIα CTD. First, we exchanged the CTDs of rat topo IIα (amino acids 1,192-1,528) and β (1,201-1,614) and examined the two chimeras' in vitro catalytic activities. Interestingly, the relaxation activities of topo IIα WT enzyme and both of the CTD-swapped mutants were inhibited in the presence of isolated cellular RNA, suggesting that the α CTD is involved in the RNA-mediated regulation of catalytic activity in topo IIα. The results of on-bead assays using a CTD-deleted mutant of rat topo IIα indicated that the RNA-mediated inhibition of the relaxation activity was caused by an interaction between the α CTD and RNA. Further, to identify the domain within the CTD that is associated with subnuclear localization of rat topo IIα, we transiently expressed EGFP-tagged CTD deletion mutants in human cells. The data indicated that the 1,192-1,289 region of rat topo IIα was required for targeting the enzyme to nucleoli. Finally, a relaxation assay using 1-1,289 and Δ1,192-1,289 truncated mutants indicated that the 1,192-1,289 region is involved in RNA-mediated inhibition. These results indicated that the CTD of rat topo IIα, containing the 1,192-1,289 region, is involved in the regulation of catalytic activity by associating with RNA, as well as in the localization to nucleoli in interphase cells.

Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions.

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.