A Conserved Residue in the Yeast Bem1p SH3 Domain Maintains the High Level of Binding Specificity Required for Function

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of “negative” interactions in determining the specificity of a domain.

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Transposon-Disruption of a Maize Nuclear Gene, tha1, Encoding a Chloroplast SecA Homologue: In Vivo Role of cp-SecA in Thylakoid Protein Targeting

Genetics ◽

10.1093/genetics/145.2.467 ◽

1997 ◽

Vol 145 (2) ◽

pp. 467-478 ◽

Cited By ~ 4

Author(s):

Rodger Voelker ◽

Janet Mendel-Hartvig ◽

Alice Barkan

Keyword(s):

Protein Targeting ◽

Sequence Similarity ◽

Nuclear Gene ◽

Cytochrome F ◽

Transposon Insertion ◽

Nuclear Mutant ◽

Strong Sequence Similarity

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced ≥40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

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The role of Fc–FcγR interactions in IgG-mediated microbial neutralization

Journal of Experimental Medicine ◽

10.1084/jem.20151267 ◽

2015 ◽

Vol 212 (9) ◽

pp. 1361-1369 ◽

Cited By ~ 92

Author(s):

Stylianos Bournazos ◽

David J. DiLillo ◽

Jeffrey V. Ravetch

Keyword(s):

Crucial Role ◽

Binding Specificity ◽

Effector Cells ◽

Effector Functions ◽

Bifunctional Molecules

Antibodies are bifunctional molecules, containing a variable Fab domain that mediates binding specificity and a constant Fc domain that bridges antibody-coated targets with FcγR-expressing cells that mediate effector functions. Although traditional mechanisms of antibody-mediated neutralization of microbes have been largely thought to result from Fab–antigen interactions, recent studies suggest that recruitment of FcγR-expressing effector cells by antibodies is a major in vivo mechanism of antibody-mediated protection from infection. In this article, we review FcγR biology, compare mammalian FcγR families, and summarize recent evidence demonstrating the crucial role that Fc–FcγR interactions play during in vivo protection from infection.

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Identification of Genes That Contribute to the Pathogenesis of Invasive Pneumococcal Disease byIn VivoTranscriptomic Analysis

Infection and Immunity ◽

10.1128/iai.00295-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3268-3278 ◽

Cited By ~ 44

Author(s):

Abiodun D. Ogunniyi ◽

Layla K. Mahdi ◽

Claudia Trappetti ◽

Nadine Verhoeven ◽

Daphne Mermans ◽

...

Keyword(s):

Invasive Pneumococcal Disease ◽

Pneumococcal Disease ◽

Targeted Mutagenesis ◽

Content Type ◽

Genome Wide ◽

Blood Relative ◽

High Level ◽

Upregulated Genes

ABSTRACTStreptococcus pneumoniae(the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-widein vivotranscriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliAand ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiOand ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products ofaliA,malX, andpiuAare promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such asS. pyogenes,Haemophilus influenzaetype b, andNeisseria meningitidis.

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The role of BH3-only protein Bim extends beyond inhibiting Bcl-2–like prosurvival proteins

The Journal of Cell Biology ◽

10.1083/jcb.200905153 ◽

2009 ◽

Vol 186 (3) ◽

pp. 355-362 ◽

Cited By ~ 131

Author(s):

Delphine Mérino ◽

Maybelline Giam ◽

Peter D. Hughes ◽

Owen M. Siggs ◽

Klaus Heger ◽

...

Keyword(s):

Family Members ◽

Binding Specificity ◽

In Vitro Studies ◽

Genetic Approach ◽

Activation Model ◽

Bh3 Domain ◽

Indirect Activation

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.

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Structural and functional analysis of a promoter of the human granulin/epithelin gene

Biochemical Journal ◽

10.1042/bj3190441 ◽

1996 ◽

Vol 319 (2) ◽

pp. 441-447 ◽

Cited By ~ 26

Author(s):

Vijay BHANDARI ◽

Rachael DANIEL ◽

Pheng Siew LIM ◽

Andrew BATEMAN

Keyword(s):

Promoter Activity ◽

Biologically Active ◽

Gene Products ◽

High Level Expression ◽

Haematopoietic Cells ◽

Molecular Masses ◽

Chloramphenicol Acetyltransferase Gene ◽

High Level

Granulins (grns) or epithelins (epis) are peptides with molecular masses of approx. 6 kDa that modulate the growth of cells. The precursor for the grns/epis, which might itself be biologically active, is a secreted glycoprotein containing multiple repeats of the grn/epi motif. Grn/epi mRNA occurs widely in vivo, particularly in tissues rich in epithelial and haematopoietic cells. To understand better the role of the gene products for grn/epi it is important to determine the patterns of grn/epi gene expression and how this is regulated. To assist in this we have obtained the 5´ sequence of the human grn/epi gene, and using chimaeras of the grn/epi -5´ sequence and the chloramphenicol acetyltransferase gene we have shown a strong promoter activity associated with the 5´ sequence of the human grn/epi gene. We have further delineated regions of the 5´ sequence that confer high-level expression on the chimaeric gene.

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Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

Journal of Virology ◽

10.1128/jvi.75.23.11284-11291.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11284-11291 ◽

Cited By ~ 161

Author(s):

David A. Einfeld ◽

Rosanna Schroeder ◽

Peter W. Roelvink ◽

Alena Lizonova ◽

C. Richter King ◽

...

Keyword(s):

Adenovirus Type ◽

Wild Type ◽

Base Modification ◽

Model Following ◽

Vector Distribution ◽

High Level ◽

Adenovirus Type 5

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

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Consequences of Phosphorylation in a Mononegavirales Polymerase-Cofactor System

Journal of Virology ◽

10.1128/jvi.02180-20 ◽

2021 ◽

Author(s):

Joseph R. Gould ◽

Shihong Qiu ◽

Qiao Shang ◽

Terje Dokland ◽

Tomoaki Ogino ◽

...

Keyword(s):

Resonance Spectroscopy ◽

Reporter System ◽

Large Protein ◽

Biologically Relevant ◽

Protein Protein Interaction ◽

A Genome ◽

Vesicular Stomatitis ◽

Negative Sense ◽

Made In

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, which consists of viruses with a genome of nonsegmented negative-sense (NNS) RNA. Many insights into the molecular biology of NNS viruses were first made in VSV, which is often studied as a prototype for members of this order. Like other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and phosphoprotein (P). Recent discoveries have produced a model in which the N-terminal disordered segment of P (PNTD) coordinates the C-terminal accessory domains to produce a “compacted” L conformation. Despite this advancement, the role of the three phosphorylation sites in PNTD has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between PNTD and the L protein C-terminal domain (LCTD), we demonstrated our ability to sensitively test for changes in the interface between the two proteins. This method showed that the binding site for PNTD on LCTD is longer than was previously appreciated. We demonstrated that phosphorylation of PNTD modulates its interaction with LCTD and used a minigenome reporter system to validate the functional significance of the PNTD-LCTD interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated PNTD displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of PNTD modulates its cofactor and conformational regulatory activities with L. IMPORTANCE Polymerase-cofactor interactions like those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Despite cofactor phosphorylation being present in most of these interactions, what effect if any it has on this protein-protein interaction had not been addressed. Our study is the first to address the effects of phosphorylation on P during its interactions with L in residue-by-residue detail. As phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggest that the structural picture of L during infection might be more complex than previously appreciated.

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The Association of Aberrant Expression of FGF1 and mTOR-S6K1 in Colorectal Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.706838 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tinghui Duan ◽

Diyuan Zhou ◽

Yizhou Yao ◽

Xinyu Shao

Keyword(s):

Colorectal Cancer ◽

Malignant Neoplasms ◽

Aberrant Expression ◽

Protein Levels ◽

And Migration ◽

High Level ◽

Proliferation And Migration

Colorectal cancer (CRC) is one of the most frequent malignant neoplasms worldwide, and the effect of treatments is limited. Fibroblast growth factor 1 (FGF1) has been involved in a wide variety of several malignant diseases and takes part in the tumorigenesis of CRC. However, the function and mechanism of FGF1 in CRC remains elusive. In this study, the results indicated that FGF1 is elevated in CRC tissues and linked with poor prognosis (P < 0.001). In subgroup analysis of FGF1 in CRC, regardless of any clinic-factors except gender, high level FGF1 expression was associated with markedly shorter survival (P < 0.05). In addition, the expression of p-S6K1 and FGF1 was not associated in normal tissue (P = 0.781), but their expression was closely related in tumor tissue (P = 0.010). The oncogenic role of FGF1 was determined using in vitro and in vivo functional assays. FGF1 depletion inhibited the proliferation and migration of CRC cells in vitro and vivo. FGF1 was also significantly correlated with mTOR-S6K1 pathway on the gene and protein levels (P < 0.05). In conclusion, FGF1 acts as a tumor activator in CRC, and against FGF1 may provide a new visual field on treating CRC, especially for mTORC1-targeted resistant patients.

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Selective Activation of Chloroplast psbD Light-Responsive Promoter and psaA/B Promoter in Transplastomic Tobacco Plants Overexpressing Arabidopsis Sigma Factor AtSIG5

Protein and Peptide Letters ◽

10.2174/0929866526666191014130605 ◽

2020 ◽

Vol 27 (2) ◽

pp. 168-175 ◽

Cited By ~ 1

Author(s):

Mikio Nozoe ◽

Yuichi Tsunoyama ◽

Yoko Ishizaki ◽

Yoichi Nakahira ◽

Takashi Shiina

Keyword(s):

Reaction Center ◽

Sigma Factor ◽

Critical Role ◽

Core Proteins ◽

Photosynthesis Genes ◽

High Level ◽

Differential Transcription ◽

D2 Protein

Background: Plastid-encoded eubacterial-type RNA polymerase (PEP) plays a critical role in the transcription of photosynthesis genes in chloroplasts. Notably, some of the reaction center genes, including psaA, psaB, psbA, and psbD genes, are differentially transcribed by PEP in mature chloroplasts. However, the molecular mechanism of promoter selection in the reaction center gene transcription by PEP is not well understood. Objective: Sigma factor proteins direct promoter selection by a core PEP in chloroplasts as well as bacteria. AtSIG5 is a unique chloroplast sigma factor essential for psbD light-responsive promoter (psbD LRP) activity. To analyze the role of AtSIG5 in chloroplast transcription in more detail, we assessed the effect of AtSIG5 hyper-expression on the transcription of plastid-encoded genes in chloroplast transgenic plants. Results: The chloroplast transgenic tobacco (CpOX-AtSIG5) accumulates AtSIG5 protein at extremely high levels in chloroplasts. Due to the extremely high-level expression of recombinant AtSIG5, most PEP holoenzymes are most likely to include the recombinant AtSIG5 in the CpOXAtSIG5 chloroplasts. Thus, we can assess the promoter preference of AtSIG5 in vivo. The overexpression of AtSIG5 significantly increased the expression of psbD LRP transcripts encoding PSII reaction center D2 protein and psaA/B operon transcripts encoding PSI core proteins. Furthermore, run-on transcription analyses revealed that AtSIG5 preferentially recognizes the psaA/B promoter, as well as the psbD LRP. Moreover, we found that psbD LRP is constitutively active in CpOX-AtSIG5 plants irrespective of light and dark. Conclusion: AtSIG5 probably plays a significant role in differential transcription of reaction center genes in mature chloroplasts.

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Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7180-7190.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7180-7190 ◽

Cited By ~ 1

Author(s):

W Kaszubska ◽

R Hooft van Huijsduijnen ◽

P Ghersa ◽

A M DeRaemy-Schenk ◽

B P Chen ◽

...

Keyword(s):

Cyclic Amp ◽

Consensus Sequence ◽

Direct Interaction ◽

Nf Kappa B ◽

Independent Manner ◽

Protein Protein Interaction ◽

Responsive Element ◽

And Function

We previously reported that NF-kappa B and a complex we referred to as NF-ELAM1 play a central role in cytokine-induced expression of the E-selectin gene. In this study we identify cyclic AMP (cAMP)-independent members of the ATF family binding specifically to the NF-ELAM1 promoter element. The NF-ELAM1 element (TGACATCA) differs by a single nucleotide substitution from the cAMP-responsive element consensus sequence. We demonstrate that this sequence operates in a cAMP-independent manner to induce transcription and thus define it as a non-cAMP-responsive element (NCRE). We show that ATFa is a component of the NF-ELAM1 complex and its overexpression activates the E-selectin promoter. In addition, ATFa, ATF2, and ATF3 interact directly with NF-kappa B in vitro, linking two unrelated families of transcription factors in a novel protein-protein interaction. Furthermore, we demonstrate that the ability of overexpressed NF-kappa B to transactivate the E-selectin promoter in vivo is dependent on the NF-ELAM1 complex. Our results suggest that a direct interaction between ATFs and NF-kappa B is, at least in part, the mechanism by which these factors specifically regulate E-selectin promoter activity.

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