Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

Subcellular localization of mammalian type II membrane proteins

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

The abundance of short proteins in the mammalian proteome

Short proteins play key roles in cell signalling and other processes, but their abundance in the mammalian proteome is unknown. Current catalogues of mammalian proteins exhibit an artefactual discontinuity at a length of 100 aa, so that protein abundance peaks just above this length and falls off sharply below it. To clarify the abundance of short proteins, we identify proteins in the FANTOM collection of mouse cDNAs by analysing synonymous and nonsynonymous substitutions with the computer program CRITICA. This analysis confirms that there is no real discontinuity at length 100. Roughly 10% of mouse proteins are shorter than 100 aa, although the majority of these are variants of proteins longer than 100 aa. We identify many novel short proteins, including a dark matter'' subset containing ones that lack detectable homology to other known proteins. Translation assays confirm that some of these novel proteins can be translated and localised to the secretory pathway.

Transposon-free regions in mammalian genomes

Despite the presence of over 3 million transposons separated on average by similar to 500 bp, the human and mouse genomes each contain almost 1000 transposon-free regions (TFRs) over 10 kb in length. The majority of human TFRs correlate with orthologous TFRs in the mouse, despite the fact that most transposons are lineage specific. Many human TFRs also overlap with orthologous TFRs in the marsupial opossum, indicating that these regions have remained refractory to transposon insertion for long evolutionary periods. Over 90% of the bases covered by TFRs are noncoding, much of which is not highly conserved. Most TFRs are not associated with unusual nucleotide composition, but are significantly associated with genes encoding developmental regulators, suggesting that they represent extended regions of regulatory information that are largely unable to tolerate insertions, a conclusion difficult to reconcile with current conceptions of gene regulation.

Electrolyte transport in the mammalian colon: mechanisms and implications for disease Kunzelmann and Marcus Mall

Electrolyte Transport in the Mammalian Colon: Mechanisms and Implications for Disease. Physiol. Rev. 82: 245-289, 2002.The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na+ channels (ENaC), the Na+-K+-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na+ feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl- secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl- channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl- secretion and enhanced Na+ absorption in the colon of cystic fibrosis (CF) patients. Ca2+- and cAMP-activated basolateral K+ channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.

Evaluation and comparison of mammalian subcellular localization prediction methods

Background: Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results: In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, and lysosome). The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion: No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE dataset and variable performance on individual subcellular localizations was observed. Proteins localized to the secretory pathway were the most difficult to predict, while nuclear and extracellular proteins were predicted with the highest sensitivity.