Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

Interactions of nucleolin and ribosomal protein L26 (RPL26) in translational control of human p53 mRNA

Ribosomal protein RPL26 enhances p53 translation after DNA damage, and this regulation depends upon interactions between the 5'- and 3'-UTRs of human p53 mRNA (Takagi, M., Absalon, M. J., McLure, K. G., and Kastan, M. B. (2005) Cell 123, 49-63; Chen, J., and Kastan, M. B. (2010) Genes Dev. 24, 2146-2156). In contrast, nucleolin (NCL) suppresses the translation of p53 mRNA and its induction after DNA damage. We confirmed reports that RPL26 and NCL interact with each other and then explored the potential role of this interaction in the translational control of p53 after stress. NCL repression of p53 translation utilizes both the 5'- and 3'-UTRs of p53 mRNA, and NCL binds to the same 5'-3'-UTR interaction region that is critical for the recruitment of RPL26 to p53 mRNA after DNA damage. We also found that NCL is able to oligomerize, consistent with a model in which NCL stabilizes this double-stranded RNA structure. We found that the RNA-binding domain of NCL participates in binding to p53 mRNA, is required for both NCL dimerization and NCL-mediated translational repression, and is the domain of NCL that interacts with RPL26. Excessive RPL26 disrupts NCL dimerization, and point mutations in the NCL-interacting region of RPL26 reduce NCL-RPL26 interactions and attenuate both RPL26 binding to human p53 mRNA and p53 induction by RPL26. These observations suggest a model in which the base pairings in the p53 UTR interaction regions are critical for both translational repression and stress induction of p53 by NCL and RPL26, respectively, and that disruption of a NCL-NCL homodimer by RPL26 may be the switch between translational repression and activation after stress.     

CFTR–SLC26 transporter interactions in epithelia

Transport mechanisms that mediate the movements of anions must be coordinated tightly in order to respond appropriately to physiological stimuli. This process is of paramount importance in the function of diverse epithelial tissues of the body, such as, for example, the exocrine pancreatic duct and the airway epithelia. Disruption of any of the finely tuned components underlying the transport of anions such as Cl⁻, HCO₃ ⁻, SCN⁻, and I⁻ may contribute to a plethora of disease conditions. In many anion-secreting epithelia, the interactions between the cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) transporters determine the final exit of anions across the apical membrane and into the luminal compartment. The molecular identification of CFTR and many SLC26 members has enabled the acquisition of progressively more detailed structural information about these transport molecules. Studies employing a vast array of increasingly sophisticated approaches have culminated in a current working model which places these key players within an interactive complex, thereby setting the stage for future work.     

The effect of neuronal expression of heat shock proteins 26 and 27 on lifespan, neurodegeneration, and apoptosis in Drosophila

Heat shock proteins (Hsps) are chaperones thought to increase lifespan, enhance stress resistance, and prevent apoptosis and neurodegenerative diseases. Our previous study reported that ubiquitous expression of hsp26 or hsp27 extended Drosophila lifespan. The effect of neuronal expression of hsp26 and hsp27 in Drosophila on the above-mentioned functions has not yet been investigated. Here, we show that neuronal expression of hsp26 or hsp27 improved lifespan and increased resistance to oxidative stress. However, only neuronal expression of hsp27 ameliorated Parkinsonism climbing disorder and attenuated mild polyglutamine-induced toxicity. Additionally, neuronal expression of hsp27 specifically partially rescued hid-induced lethality, but was not able to rescue reaper/grim-induced lethality. However, unlike hsp27, neuronal expression of hsp26 did not rescue hid-induced or reaper/grim-induced lethality. In summary, we demonstrate the functional similarities and differences of neuronal expression of hsp26 and hsp27 in adult Drosophila.     

Ribosomal Protein Binding with the First Intron of the Human rpS26 Pre-mRNA Stimulates Its Interaction with Proteins Extracted from HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, proteins of the 40S ribosome subunit bind to the first intron of the rpS26 pre-mRNA. The binding involved mostly S23, S26 and, to a lesser extent, S13/16. Negligible binding was observed for S2/3a, S6, S8, S10, S11, and S20. Small-subunit proteins did not affect the efficiency of in vitro splicing of a pre-mRNA fragment corresponding to the first intron, second exon, second intron, and a part of the third exon of the rpS26 gene. However, ribosomal proteins substantially increased UV-induced adduction of the pre-mRNA fragments with nuclear extract proteins of HeLa cells. The same set of HeLa proteins was observed with each pre-mRNA fragment. Ribosomal proteins formed adducts only in the absence of HeLa proteins.     

MOLECULAR PHYLOGENETIC RELATIONSHIPS IN THE FRESHWATER FAMILY HYDRODICTYACEAE (SPHAEROPLEALES,CHLOROPHYCEAE), WITH AN EMPHASIS ON PEDIASTRUM DUPLEX1

The freshwater green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyta) has traditionally consisted of four coenobial genera, Pediastrum Meyen 1829, Hydrodictyon Roth 1797, Sorastrum Kützing 1845, and Euastropsis Lagerheim1894. Two recent molecular phylogenetic studies demonstrated the need for reevaluation of the generic and species boundaries in this morphology‐rich family. This study expands the previous work to include phylogenetic analyses of 103 ingroup isolates representing North America, Europe, and Australia, with an emphasis on the common and geographically widespread species Pediastrum duplex. Nucleotide sequence data were collected from the nuclear LSU (26S rDNA) and the chloroplast RUBISCO LSU (rbcL) genes, totaling >3,000 aligned characters. The 26S and rbcL data sets were analyzed using maximum‐likelihood (ML) and Bayesian phylogenetic methods. In addition, SEM was used to examine the wall morphology of a majority of the isolates. The results supported previous indications that the P. duplex Meyen 1829 morphotype is nonmonophyletic and resolved some previously ambiguous relationships recovered in earlier phylogenetic estimations using fewer isolates. These new data allowed testing of the recent taxonomic revisions of the family that split Pediastrum into five genera. Some of the previous revisions by Buchheim et al. (2005) were well supported (erection of Stauridium and Monactinus), while others were not (Pediastrum, Pseudopediastrum, Parapediastrum).     

STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro

There is widespread interest in the use of bone marrow stromal cells (BMSC) for tissue reconstruction and repair and for gene therapy. BMSC represent the differentiated progeny of CFU-F, which however comprise a developmentally heterogeneous population as is reflected in the cellular heterogeneity of the cell populations to which they give rise. We have compared the efficacy of monoclonal antibodies recognising a series of stromal antigens, viz. STRO-1, HOP-26, CD49a and SB-10/CD166, as tools for the enrichment of CFU-F prior to culture and as developmental markers for culture-expanded BMSC. In freshly isolated bone marrow mononuclear cells (BMMNC), the proportion of antigen-positive cells was 27%, 46%, 5% and 19% for STRO-1, HOP-26, CD49a and CD166, respectively. All CD49a⁺ cells co-expressed STRO-1. The degree of CFU-F enrichment obtained with anti-CD49a (~18-fold) by a one-pass immunoselection strategy was significantly greater than that of all other antibodies tested. BMSC expressed higher levels of all antigens investigated (except for HOP-26) compared with BMMNC. Expression of STRO-1 and CD49a remained restricted to a subset of BMSC, whereas all BMSC were SB-10/CD166 positive. Treatment with dexamethasone (10 nM), which promotes the differentiation and further maturation of cells of the osteogenic lineage in this cell culture system, increased the expression of CD49a and HOP-26. The CD49a⁺ and HOP-26⁺ fractions of BMSC were further subdivided by dual-labelling with anti-STRO-1 and B4–78 (an antibody recognising the B/L/K isoform of the enzyme alkaline phosphatase), respectively. By using a variety of criteria, the HOP-26 antigen was identified as CD63, a member of the tetraspanin family of proteins thought to modulate integrin compartmentalisation and signalling.K.S., S.W., C.M.J. and J.A.L. gratefully acknowledge the financial support of the University Bath, the Arthritis Research Campaign and the Wellcome Trust     

Essential function of the N‐termini tails of the proteasome for the gating mechanism revealed by molecular dynamics simulations

Proteasome is involved in the degradation of proteins. Proteasome activators bind to the proteasome core particle (CP) and facilitate opening a gate of the CP, where Tyr8 and Asp9 in the N‐termini tails of the CP form the ordered open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N‐termini tails are disordered and the stabilized open‐gate conformation cannot be formed. To understand the gating mechanism of the CP for the translocation of the substrate, four different molecular dynamics simulations were carried out: ordered‐ and Tyr8Gly/Asp9Gly disordered‐gate models of the CP complexed with an ATP‐independent PA26 and ordered‐ and disordered‐gate models of the CP complexed with an ATP‐dependent PAN‐like activator. The free‐energies of the translocation of a polypeptide substrate moving through the gate were estimated. In the ordered‐gate models, the substrate in the activator was more stable than that in the CP. The conformational entropy of the N‐termini tails of the CP was larger when the substrate was in the activator than in the CP. In the disordered‐gate models, the substrate in the activator was more destabilized than in the ordered‐gate models. The mutated N‐termini tails became randomized and their increased conformational entropy could no longer increase further even when the substrate was in the activator, meaning the randomized N‐termini tails had lost the ability to stabilize the substrate in the activator. Thus, it was concluded that the dynamics of the N‐termini tails entropically play a key role in the translocation of the substrate. Proteins 2014; 82:1985–1999. © 2014 Wiley Periodicals, Inc.     

Proteasomes Can Degrade a Significant Proportion of Cellular Proteins Independent of Ubiquitination

The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSs) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably well conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSs at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.