Recognition Subunit Research Articles

Phase Variation of Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes phase variation or spontaneous, reversible phenotypic variation in colony opacity, encapsulation, and pilus expression. The variation in colony opacity appears to occur in all strains, whereas the switches in the production of the capsule and pilus have been observed in several strains. This chapter elaborates on the variation in colony opacity since this phenomenon has been extensively characterized. S. pneumoniae produces opaque and transparent colonies on the translucent agar medium. The different colony phases are fundamentally distinct phenotypes in their metabolism and multiple characteristics, as exemplified by cell surface features and phenotypes in colonization and virulence. Opaque variants, which express more capsular polysaccharides and fewer teichoic acids, are more virulent in animal models of sepsis but colonize the nasopharynx poorly. In contrast, transparent variants, with fewer capsular polysaccharides and more teichoic acid, colonize the nasopharynx in animal models more efficiently but are relatively avirulent. Lastly, pneumococcal opacity variants are generated by differential methylation of the genome DNA variation. The reversible switch in the methylation pattern is caused by DNA inversions in three homologous hsdS genes of the colony opacity determinant (cod) or SpnD39III locus, a conserved type I restriction-modification (RM) system. The hsdS gene encodes the sequence recognition subunit of the type I RM DNA methyltransferase. The combination of DNA inversion and differential methylation, a complex mechanism of phase variation, generates a mixed population that may allow for the selection of organisms in vivo with characteristics permissive for either carriage or systemic infection.

The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response.

FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.

Cyclin F-Dependent Degradation of RBPJ Inhibits IDH1R132H-Mediated Tumorigenesis.

Cyclin F is a substrate recognition subunit of Skp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase complex. Although there have been reports describing the role of cyclin F in the genotoxic stress response, its function under conditions of altered metabolic homeostasis remain unexplored. Here we report that cyclin F is induced upon metabolic stress in a FOXO1-dependent manner. Under metabolic stress conditions, cyclin F mediated polyubiquitylation of RBPJ at Lys315, leading to its proteasomal degradation. RBPJ regulated the expression of IDH1, which is often mutated to an oncogenic form IDH1R132H in cancers. Thus, metabolic stress-induced cyclin F attenuated the oncogenic functions of IDH1R132H in an RBPJ-dependent manner. Studies in mouse tumor models indicated that abrogation of cyclin F expression facilitates IDH1R132H-mediated tumorigenesis and metastasis. In addition, increased IDH1R132H levels correlated with reduced cyclin F levels in increasing grades of glioma. These findings highlight a novel aspect of cyclin F functions in inhibiting tumorigenesis and provide mechanistic insights into regulation of IDH1R132H Significance: These findings reveal mechanistic insights into the key role of the cyclin F-RBPJ axis in response to metabolic stress in cancer cells. Cancer Res; 78(22); 6386-98. ©2018 AACR.

The ubiquitin ligase subunit β-TrCP in Sertoli cells is essential for spermatogenesis in mice

β-TrCP is the substrate recognition subunit of an SCF-type ubiquitin ligase. We recently showed that deletion of the genes for both β-TrCP1 and β-TrCP2 paralogs in germ cells of male mice resulted in accumulation of the transcription factor DMRT1 and spermatogenic failure, whereas systemic β-TrCP1 knockout combined with β-TrCP2 knockdown had previously been shown to lead to disruption of testicular organization and accumulation of the transcription factor SNAIL. Here we investigated β-TrCP function in Sertoli cells by generating mice with targeted deletion of the β-TrCP2 gene in Sertoli cells on a background of whole-body β-TrCP1 knockout. Loss of β-TrCP in Sertoli cells caused infertility due to a reduction in the number of mature sperm. Whereas spermatogonia were not affected, male germ cells entered meiosis prematurely and the number of round spermatids was reduced in the mutant mice. Extracts of Sertoli cells and of the testis from the mutant mice manifested accumulation of SNAIL, and expression of the SNAIL target gene for E-cadherin was down-regulated in Sertoli cells from these animals. Our results indicate that β-TrCP in Sertoli cells regulates Sertoli cell–germ cell interaction through degradation of SNAIL, with such regulation being critical for sperm development.

Controlling Structure Beyond the Initial Coordination Sphere: Complexation-Induced Reversed Micelle Formation in Calix[4]pyrrole-Containing Diblock Copolymers.

A diblock copolymer containing a strapped calix[4]pyrrole-based ion pair recognition subunit has been synthesized via RAFT polymerization. As prepared, the polymer is hydrophobic and devoid of any particular morphological form. However, upon ion pair complexation, the copolymer self-assembles to generate reverse micelles in organic media. The reverse micelles formed in this way may be used to extract alkali cation and cesium halide anion salts from an aqueous source into an organic receiving phase. The polymer proved more effective as an extractant than the corresponding free ion pair receptor.

Regulation of the Gid ubiquitin ligase recognition subunit Gid4.

Glucose consumption via glycolysis and its biosynthesis via gluconeogenesis are central reciprocal pathways controlled by a set of different enzymes. In the yeast Saccharomycescerevisiae, expression of gluconeogenic enzymes is induced when cells are devoid of glucose. Availability of glucose immediately leads to inactivation and rapid degradation of these enzymes via the ubiquitin proteasome system. Polyubiquitination is carried out by the Gid complex, a multisubunit RING E3 ligase that constitutively consists of six different proteins. Upon addition of glucose to the medium, the substrate recognition subunit Gid4 appears within minutes and triggers ubiquitination of the gluconeogenic enzymes. Here, we show that Gid4 is tightly regulated on the transcriptional and protein level to ensure proper adjustment of gluconeogenesis.

Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.

New bifunctional restriction-modification enzyme AloI isoschizomer (PcoI): Bioinformatics analysis, purification and activity confirmation

Type II restriction endonucleases and modification DNA-methyltransferases are key instruments of genetic engineering. Recently the number of proteins assigned to this group exceeds 8500. Subtype IIC organizes bifunctional endonuclease-methyltransferase enzymes and currently consists of 16 described members. Here we present phylogenetic tree of 22 new potential bifunctional endonucleases. The majority of them are thought to be fusions of a restriction nuclease with a DNA-methyltransferase and a target recognition subunit of type I restriction-modification systems (R-M-S structure). A RM.AloI isoschizomer from Prevotella copri DSM-18205, PcoI, has been cloned, purified and its REase activity demonstrated. It cuts DNA in magnesium-dependent manner and demonstrates high affinity to DNA, which probably reflects its mechanism of action. This work provides additional proves that gene fusion might play an important role in evolution of restriction-modification systems and other DNA-modifying proteins.

The von Hippel–Lindau Tumor Suppressor Protein

The von Hippel–Lindau (VHL) gene is a two-hit tumor suppressor gene and is linked to the development of the most common form of kidney cancer, clear cell renal carcinoma; blood vessel tumors of the retina, cerebellum, and spinal cord called hemangioblastomas; and tumors of the sympathoadrenal nervous system called paragangliomas. The VHL gene product, pVHL, is the substrate recognition subunit of a cullin-dependent ubiquitin ligase that targets the α subunits of hypoxia-inducible factor (HIF) for destruction when oxygen is plentiful. Mounting evidence implicates HIF2 in the pathogenesis of pVHL-defective tumors and has provided a conceptual foundation for the development of drugs to treat them that inhibit HIF2-responsive gene products such as VEGF and, more recently, HIF2 itself. pVHL has additional, noncanonical functions that are cancer relevant, including roles related to the primary cilium, chromosome stability, extracellular matrix formation, and survival signaling.

MAPK signaling couples SCF-mediated degradation of translational regulators to oocyte meiotic progression

RNA-binding proteins (RBPs) are important regulators of gene expression programs, especially during gametogenesis. How the abundance of particular RBPs is restricted to defined stages of meiosis remains largely elusive. Here, we report a molecular pathway that subjects two nonrelated but broadly evolutionarily conserved translational regulators (CPB-3/CPEB and GLD-1/STAR) to proteosomal degradation in Caenorhabditis elegans germ cells at the transition from pachytene to diplotene of meiotic prophase. Both RBPs are recognized by the same ubiquitin ligase complex, containing the molecular scaffold Cullin-1 and the tumor suppressor SEL-10/FBXW7 as its substrate recognition subunit. Destabilization of either RBP through this Skp, Cullin, F-box-containing complex (SCF) ubiquitin ligase appears to loosen its negative control over established target mRNAs, and presumably depends on a prior phosphorylation of CPB-3 and GLD-1 by MAPK (MPK-1), whose activity increases in mid- to late pachytene to promote meiotic progression and oocyte differentiation. Thus, we propose that the orchestrated degradation of RBPs via MAPK-signaling cascades during germ cell development may act to synchronize meiotic with sexual differentiation gene expression changes.

Refining Radiation for the Next Century.

![][1] Wilhelm Roentgen's report more than a century ago describing X-ray imaging fostered in the application of ionizing radiation (IR) for cancer therapy. Today, about half of all the patients with cancer are treated with radiotherapy and it remains one of our most effective

BAF180: Its Roles in DNA Repair and Consequences in Cancer.

In 2011, Varela et al. reported that the PBRM1 gene is mutated in approximately 40% of clear cell renal cell carcinoma cases. Since then, the number of studies relating PBRM1 mutations to cancers has substantially increased. BAF180 has now been linked to more than 30 types of cancers, including ccRCC, cholangiocarcinomas, esophageal squamous cell carcinoma, bladder cancer, and breast cancer. The mutations associated with BAF180 are most often truncations, which result in a loss of protein expression. This loss has been shown to adversely affect the expression of genes, likely because BAF180 is the chromatin recognition subunit of PBAF. In addition, BAF180 functions in numerous DNA repair mechanisms. Its roles in mediating DNA repair are likely the mechanism by which BAF180 acts a tumor suppressor protein. As research on this protein gains more interest, scientists will begin to piece together the complicated puzzle of the BAF180 protein and why its loss often results in cancer.

Novel Immunotherapy Agents for Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) in adults has a survival rate of 40–50% at 5 years, with a high relapse rate after first-line chemotherapy. After relapse, results with salvage therapy are currently unsatisfactory. Therefore, both the optimisation of front-line therapy to reduce relapse incidence and the search for effective salvage therapies for relapsed/refractory (r/r) ALL have been of great interest to the medical community in recent years. The well-characterised expression of well-defined cell-surface antigens in B cell ALL (B)-ALL and T cell (T)-ALL, such as CD19, CD20, CD22, and CD52, has led to the development of several immunotherapy strategies, comprising ‘nude’ monoclonal antibodies (moAbs), conjugated moAbs, bispeciphic, or highly sophisticated chimeric antigen receptor (CAR)-T cell therapy. Recently, both the bispecific moAb blinatumomab (anti-CD19 coupled with a CD3 recognition subunit) and the conjugated anti-CD22 moAb inotuzumab-ozogamicin have resulted in higher remission rates (44% versus 25%, and 80.7% versus 29.4%, respectively) and survival advantages (median overall survival [OS]: 7.7 months versus 4 months, and 7.7 months versus 6.7 months, respectively) in patients with r/r B-ALL when compared to standard salvage chemotherapy-based regimens. On the other hand, preliminary reports show feasibility and unprecedented response rates of ≤90% in highly refractory children and adults treated with CAR-modified T cells targeting the B cell specific CD19 antigen, which seem to be durable in a significant proportion of patients. Furthermore, the addition of anti-CD20 moAb rituximab to front-line standard chemotherapy in patients with CD20+ B-ALL has resulted in a clinical benefit, with prolongation of response duration and survival (3-year leukaemia-free survival and OS: 70% versus 38%; p<0.001, and 75% versus 47%; p=0.003). In conclusion, immunotherapy is currently providing additional options for high-risk ALL patients both in front-line or advanced phase. Nonetheless, the optimal positioning of these novel agents, specially in relation to allogeneic haematopoietic stem-cell transplantion, needs to be clarified. This article aims to review several of these new therapeutic immunotherapy options available for patients with adult ALL, as well as their specific toxicity profile.

β-transducin repeat-containing E3 ubiquitin protein ligase inhibits migration, invasion and proliferation of glioma cells.

β-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) serves as the substrate recognition subunit for the Skp1-Cullin1-F-box protein E3 ubiquitin ligase, which recognizes the double phosphorylated DSG (X)2+nS destruction motif in various substrates that are essential for numerous aspects of tumorigenesis and regulates several important signaling pathways. However, the biological significance of β-TrCP in glioma progression remains largely unknown. A previous study by the authors demonstrated that the levels of β-TrCP protein expression in brain glioma tissues were significantly lower compared with non-tumorous tissues and that higher grades of gliomas exhibited lower levels of β-TrCP expression in comparison with lower glioma grades. In addition, low β-TrCP expression was associated with poor prognosis in patients with glioma. Subsequently, the present study aimed to investigate the effect of β-TrCP on migratory, invasive and proliferative abilities of glioma cells. β-TrCP plasmids were transfected into cultured U251 and U87 glioma cells, and changes in migration, invasion and proliferation were analyzed using wound healing, Transwell and EdU assays. It was identified that the overexpression of β-TrCP inhibited migration, invasion and proliferation in glioma cells. In summary, these results indicate that β-TrCP may serve a protective role against the progression of glioma by suppressing cell migration, invasion and proliferation. The potential mechanism of β-TrCP I glioma cells requires additional investigation.

A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation

Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate.IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.

The evolution and functional characterization of miiuy croaker interferon regulatory factor 9 involved in immune response

Interferon regulatory factors (IRFs) are transcription factors which play important roles in regulating the expression of type I interferons (IFNs) and IFN-stimulated genes. IRF9 is one of the IRF family gene members which belongs to the IRF4 subfamily. Mammalian IRF9 has been known to be involved in antiviral responses as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex. In fish, only a few studies investigated the characteristics of IRF9 and the role in IFN signaling. In this study, we identified the IRF9 gene from miiuy croaker (mmiIRF9) and studied its feature and function. Sequence analysis showed the similarity of mmiIRF9 and other fish IRF9 genes. Structural and syntenic analysis showed the conservatism in fish IRF9 genes. The result of expression analysis in normal tissues and infected tissues and macrophages showed that mmiIRF9 expressed in all tested normal tissues and up-regulated expression in liver, kidney and macrophages after stimulated with poly(I:C). Luciferase reporter assays demonstrated the mmiIRF9 can induced IFNα and IFNβ luciferase reporters and the cellular localization of mmiIRF9 was mainly distributed in the cytoplasm in Hela cells. Furthermore, the evolutionary analysis of IRF4 subfamily showed the IRF4 and IRF8 may be the most ancient and conservative genes in the evolution of this subfamily.

Physical and Functional Characterization of a Viral Genome Maturation Complex

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Cockayne syndrome B protein regulates recruitment of the Elongin A ubiquitin ligase to sites of DNA damage

Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.

FBXL5 Inactivation in Mouse Brain Induces Aberrant Proliferation of Neural Stem Progenitor Cells.

FBXL5 is the substrate recognition subunit of an SCF-type ubiquitin ligase that serves as a master regulator of iron metabolism in mammalian cells. We previously showed that mice with systemic deficiency of FBXL5 fail to sense intracellular iron levels and die in utero at embryonic day 8.5 (E8.5) as a result of iron overload and subsequent oxidative stress. This early embryonic mortality has thus impeded study of the role of FBXL5 in brain development. We have now generated mice lacking FBXL5 specifically in nestin-expressing neural stem progenitor cells (NSPCs) in the brain. Unexpectedly, the mutant embryos manifested a progressive increase in the number of NSPCs and astroglia in the cerebral cortex. Stabilization of iron regulatory protein 2 (IRP2) as a result of FBXL5 deficiency led to accumulation of ferrous and ferric iron as well as to generation of reactive oxygen species. Pharmacological manipulation suggested that the phenotypes of FBXL5 deficiency are attributable to aberrant activation of mammalian target of rapamycin (mTOR) signaling. Our results thus show that FBXL5 contributes to regulation of NSPC proliferation during mammalian brain development.

Pseudophosphatase as E3 ubiquitin ligase inhibitor.

The pseudophosphatase STYX prevents the substrate recognition subunit FBXW7 from binding the catalytic E3 ubiquitin ligase complex.