Characterization of the Dictyostelium Homolog of Chromatin Binding Protein DET1 Suggests a Conserved Pathway Regulating Cell Type Specification and Developmental Plasticity

ABSTRACT DET1 (De-etiolated 1) is a chromatin binding protein involved in developmental regulation in both plants and animals. DET1 is largely restricted to multicellular eukaryotes, and here we report the characterization of a DET1 homolog from the social amoeba Dictyostelium discoideum . As in other species, Dictyostelium DET1 is nuclear localized. In contrast to other species, where it is an essential protein, loss of DET1 is nonlethal in Dictyostelium , although viability is significantly reduced. The phenotype of the det1 − mutant is highly pleiotropic and results in a large degree of heterogeneity in developmental parameters. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed cell type patterning with a bias toward the prestalk pathway. A number of DET1-interacting proteins are conserved in Dictyostelium , and the apparently conserved role of DET1 in regulatory pathways involving the bZIP transcription factors DimB, c-Jun, and HY5 suggests a highly conserved mechanism regulating development in multicellular eukaryotes. While the mechanism by which DET1 functions is unclear, it appears that it has a key role in regulation of developmental plasticity and integration of information on environmental conditions into the developmental program of an organism.

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Characterization of a Gly19 → Val mutant of ram p25, a low Mr GTP-binding protein: Loss of GTP/GDP-binding activity in the mutated ram p25

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(92)91562-5 ◽

1992 ◽

Vol 189 (1) ◽

pp. 330-335 ◽

Cited By ~ 2

Author(s):

Koh-ichi Nagata ◽

Takeshi Suzuki ◽

Yukio Okano ◽

Michinari Hamaguchi ◽

Yoshinori Nozawa

Keyword(s):

Binding Protein ◽

Binding Activity ◽

Protein Loss ◽

Gtp Binding Protein ◽

Gtp Binding

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Characterization of Intracellular Growth RegulatoricgRby Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

Infection and Immunity ◽

10.1128/iai.00537-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3068-3076 ◽

Cited By ~ 8

Author(s):

Carolyn R. Morris ◽

Christen L. Grassel ◽

Julia C. Redman ◽

Jason W. Sahl ◽

Eileen M. Barry ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Diarrheal Disease ◽

Intracellular Growth ◽

Bioinformatics Analyses ◽

Content Type ◽

Regulatory Pathways

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods within vitroandin vivoassays to provide new insights into pathogenesis. Comparisons ofin vivoandin vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present inS. flexneriisolates but not in the three otherShigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific toS. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designatedicgR, forintracellulargrowthregulator. Deletion oficgRcaused no difference in growthin vitrobut resulted in increased intracellular replication in HCT-8 cells. Furtherin vitroandin vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown inShigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

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Characterization of Neisseria meningitidis Isolates That Do Not Express the Virulence Factor and Vaccine Antigen Factor H Binding Protein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00055-11 ◽

2011 ◽

Vol 18 (6) ◽

pp. 1002-1014 ◽

Cited By ~ 65

Author(s):

Jay Lucidarme ◽

Lionel Tan ◽

Rachel M. Exley ◽

Jamie Findlow ◽

Ray Borrow ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Binding Protein ◽

Factor H ◽

Negative Regulator ◽

Vaccine Antigen ◽

Natural Immunity ◽

Content Type ◽

Reading Frame ◽

Flanking Sequences

ABSTRACTNeisseria meningitidisremains a leading cause of bacterial sepsis and meningitis. Complement is a key component of natural immunity against this important human pathogen, which has evolved multiple mechanisms to evade complement-mediated lysis. One approach adopted by the meningococcus is to recruit a human negative regulator of the complement system, factor H (fH), to its surface via a lipoprotein, factor H binding protein (fHbp). Additionally, fHbp is a key antigen in vaccines currently being evaluated in clinical trials. Here we characterize strains ofN. meningitidisfrom several distinct clonal complexes which do not express fHbp; all strains were recovered from patients with disseminated meningococcal disease. We demonstrate that these strains have either a frameshift mutation in thefHbpopen reading frame or have entirely lostfHbpand some flanking sequences. No fH binding was detected to other ligands among thefHbp-negative strains. The implications of these findings for meningococcal pathogenesis and prevention are discussed.

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Structural Characterization of the Erythrocyte Binding Domain of the Reticulocyte Binding Protein Homologue Family of Plasmodium yoelii

Infection and Immunity ◽

10.1128/iai.01326-10 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2880-2888 ◽

Cited By ~ 11

Author(s):

Ardina Grüber ◽

Karthigayan Gunalan ◽

Jeya Kumar Ramalingam ◽

Malathy S. S. Manimekalai ◽

Gerhard Grüber ◽

...

Keyword(s):

Binding Protein ◽

Intervention Strategy ◽

Plasmodium Yoelii ◽

Binding Domain ◽

Content Type ◽

Binding Domains ◽

X Ray Scattering ◽

Ligand Interactions ◽

Erythrocyte Binding

ABSTRACTInvasion of the host cell by the malaria parasite is a key step for parasite survival and the only stage of its life cycle where the parasite is extracellular, and it is therefore a target for an antimalaria intervention strategy. Multiple members of the reticulocyte binding protein homologues (RH) family are found in all plasmodia and have been shown to bind to host red blood cells directly. In the study described here, we delineated the erythrocyte binding domain (EBD) of one member of the RH family, termed Py235, fromPlasmodium yoelii. Moreover, we have obtained the low-resolution structure of the EBD using small-angle X-ray scattering. Comparison of the EDB structure to other characterizedPlasmodiumreceptor binding domains suggests that there may be an overall structural conservation. These findings may help in developing new approaches to target receptor ligand interactions mediated by parasite proteins.

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Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

Journal of Virology ◽

10.1128/jvi.02270-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11681-11691 ◽

Cited By ~ 28

Author(s):

Emmanuelle R. J. Quemin ◽

Maija K. Pietilä ◽

Hanna M. Oksanen ◽

Patrick Forterre ◽

W. Irene C. Rijpstra ◽

...

Keyword(s):

Dna Binding ◽

Biochemical Characterization ◽

Binding Protein ◽

Structural Data ◽

Dna Binding Protein ◽

Ecological Niches ◽

Chromatin Protein ◽

Content Type ◽

Cellular Origin

ABSTRACTGeothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive toArchaeaand belong to two distinct viral families. The larger of the two families, theFuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages.Sulfolobusspindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions inArchaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain theSulfolobus solfataricuschromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress.IMPORTANCEAlthough spindle-shaped viruses represent one of the most prominent viral groups inArchaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions.

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Microtiter Plate-Based Assay for Inhibitors of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01327-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2783-2787 ◽

Cited By ~ 8

Author(s):

Sudheer Bobba ◽

V. K. Chaithanya Ponnaluri ◽

Mridul Mukherji ◽

William G. Gutheil

Keyword(s):

Staphylococcus Aureus ◽

Binding Protein ◽

Binding Constants ◽

Public Health Problem ◽

Microtiter Plate ◽

Penicillin Binding Protein ◽

Methicillin Resistant ◽

Content Type ◽

High Level

ABSTRACTPenicillin-binding protein 2a (PBP2a), the molecular determinant for high-level β-lactam resistance in methicillin-resistantStaphylococcus aureus(MRSA), is intrinsically resistant to most β-lactam antibiotics. The development and characterization of new inhibitors targeting PBP2a would benefit from an effective and convenient assay for inhibitor binding. This study was directed toward the development of a fluorescently detected β-lactam binding assay for PBP2a from MRSA. Biotinylated ampicillin and biotinylated cephalexin were tested as tagging reagents for fluorescence detection by using a streptavidin-horseradish peroxidase conjugate. Both bound surprisingly well to PBP2a, with binding constants of 1.6 ± 0.4 μM and 13.6 ± 0.8 μM, respectively. Two forms of the assay were developed, a one-step direct competition form of the assay and a two-step indirect competition form of the assay, and both forms of the assay gave comparable results. This assay was then used to characterize PBP2a binding to ceftobiprole, which gave results consistent with previous studies of ceftobiprole-PBP2a binding. This assay was also demonstrated for screening for PBP2a inhibitors by screening a set of 13 randomly selected β-lactams for PBP2a inhibition at 750 μM. Meropenem was observed to give substantial inhibition in this screen, and a follow-up titration experiment determined its apparentKito be 480 ± 70 μM. The availability of convenient and sensitive microtiter-plate based assays for the screening and characterization of PBP2a inhibitors is expected to facilitate the discovery and development of new PBP2a inhibitors for use in combating the serious public health problem posed by MRSA.

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