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Special Issue "Hypothetical Proteins"

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry, Molecular Biology and Biophysics".

Deadline for manuscript submissions: closed (29 February 2012)

Special Issue Editor

Guest Editor
Prof. Dr. Annalisa Santucci

Dipartimento di Biotecnologie, Universita' degli Studi di Siena, via Fiorentina 1, 53100 Siena, Italy
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Special Issue Information

Dear Colleagues,

The development of automated genome sequencing provided researchers with a tremendous wealth of biological information, whose potential has still to be fully realized. Although often conserved among different organisms, Open Reading Frames (ORFs) encode also for ‘hypothethical proteins’ (HPs), which are defined as proteins predicted from nucleic acid sequences but whose existence has not been proved by any experimental chemical evidence. Often, HPs cannot be related to other proteins with known structure or function; such a lack of sequence similarity is one of the main constraints to any genome functional annotation strategy.

Advances in a broad spectrum of genetic and biochemical tools speeded up the genome annotation process via structural approaches. In this context, high throughput crystallization techniques, NMR spectroscopy, X-ray diffraction and structural analyses combined with “omics” technologies such as structural genomics, transcriptomics and proteomics contributed to determine the tertiary structures and the number of isoforms or molecular species of many HPs, as wells as their intracellular/extracellular location also in relation to their function. Furthermore, a great effort to understand the geometrical location of functional site and the biochemical/biological functions of proteins lay in the development of bioinformatic tools and databases. However, much effort still needs to be dedicated to the optimization of technological platforms and bioinformatic tools for the functional analysis of HPs, in order to contribute substantially to the piecing together of the structure-function puzzle.

Prof. Dr. Annalisa Santucci
Guest Editor

Keywords

  • data mining
  • functional analysis
  • genome sequencing
  • bioinformatics
  • structural genomics
  • transcriptomics
  • proteomics

Published Papers (4 papers)

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Research

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Open AccessArticle Solution NMR Structure of Hypothetical Protein CV_2116 Encoded by a Viral Prophage Element in Chromobacterium violaceum
Int. J. Mol. Sci. 2012, 13(6), 7354-7364; doi:10.3390/ijms13067354
Received: 20 April 2012 / Revised: 25 May 2012 / Accepted: 4 June 2012 / Published: 14 June 2012
PDF Full-text (1213 KB) | HTML Full-text | XML Full-text
Abstract
CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e−07) corresponding to a hypothetical protein OR16_04617 from
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CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e−07) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and 13C- and 15N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β -sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum. Full article
(This article belongs to the Special Issue Hypothetical Proteins)
Open AccessArticle Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR)
Int. J. Mol. Sci. 2012, 13(4), 4920-4936; doi:10.3390/ijms13044920
Received: 8 February 2012 / Revised: 19 March 2012 / Accepted: 20 March 2012 / Published: 19 April 2012
Cited by 4 | PDF Full-text (760 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth
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Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2)/endothelial cell-specific chemotaxis regulator (ECSCR). In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2), differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293). Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF)-induced cell migration. Taken together, we have provided several lines of experimental evidence that two mouse ECSCR splicing variants/isoform precursors exist. They are differentially expressed in a variety of tissue types and likely involved in modulation of vascular EC migration. We have also defined the gene structure of mouse ECSCR using bioinformatics tools, which provides new information towards a better understanding of alternative splicing of ECSCR. Full article
(This article belongs to the Special Issue Hypothetical Proteins)
Figures

Open AccessArticle Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578
Int. J. Mol. Sci. 2012, 13(1), 901-917; doi:10.3390/ijms13010901
Received: 27 October 2011 / Revised: 29 December 2011 / Accepted: 9 January 2012 / Published: 16 January 2012
Cited by 6 | PDF Full-text (3917 KB) | HTML Full-text | XML Full-text
Abstract
Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of
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Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity. Full article
(This article belongs to the Special Issue Hypothetical Proteins)

Review

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Open AccessReview Structural Analysis of Hypothetical Proteins from Helicobacter pylori: An Approach to Estimate Functions of Unknown or Hypothetical Proteins
Int. J. Mol. Sci. 2012, 13(6), 7109-7137; doi:10.3390/ijms13067109
Received: 9 March 2012 / Revised: 29 May 2012 / Accepted: 1 June 2012 / Published: 8 June 2012
Cited by 7 | PDF Full-text (2870 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Helicobacter pylori (H. pylori) have a unique ability to survive in extreme acidic environments and to colonize the gastric mucosa. It can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, gastric cancer, etc.
[...] Read more.
Helicobacter pylori (H. pylori) have a unique ability to survive in extreme acidic environments and to colonize the gastric mucosa. It can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, gastric cancer, etc. Based on genomic research of H. pylori, over 1600 genes have been functionally identified so far. However, H. pylori possess some genes that are uncharacterized since: (i) the gene sequences are quite new; (ii) the function of genes have not been characterized in any other bacterial systems; and (iii) sometimes, the protein that is classified into a known protein based on the sequence homology shows some functional ambiguity, which raises questions about the function of the protein produced in H. pylori. Thus, there are still a lot of genes to be biologically or biochemically characterized to understand the whole picture of gene functions in the bacteria. In this regard, knowledge on the 3D structure of a protein, especially unknown or hypothetical protein, is frequently useful to elucidate the structure-function relationship of the uncharacterized gene product. That is, a structural comparison with known proteins provides valuable information to help predict the cellular functions of hypothetical proteins. Here, we show the 3D structures of some hypothetical proteins determined by NMR spectroscopy and X-ray crystallography as a part of the structural genomics of H. pylori. In addition, we show some successful approaches of elucidating the function of unknown proteins based on their structural information. Full article
(This article belongs to the Special Issue Hypothetical Proteins)

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