bilbo, a non-LTR retrotransposon of Drosophila subobscura: a clue to the evolution of LINE-like elements in Drosophila

We used the repetitive character of transposable elements to isolate a non-LTR retrotransposon in Drosophila subobscura. bilbo, as we have called it, has homology to TRIM and LOA elements. Sequence analysis showed a 5' untranslated region (UTR), an open reading frame (ORF) with no RNA-binding domains, a downstream ORF that had structural homology to that of the I factor, and, finally, a 3' UTR which ended in several 5-nt repeats. The results of our phylogenetic and structural analyses shed light on the evolution of Drosophila non-LTR retrotransposons and support the hypothesis that an ancestor of these elements was structurally complex.      

Variation of physico-chemical parameters along a river transect through the Okavango Delta,Botswana

The Okavango Delta depends on water quantity and quality to sustain its ecosystem services. Whereas many studies have been carried out on its hydrology, few have been done on water quality in the delta. Water pH, electrical conductivity (EC), dissolved oxygen (DO), turbidity, total suspended solids (TSS) and dissolved organic carbon (DOC) were monitored at 10 sites along the Okavango–Boro–Thamalakane–Lake Ngami system almost fortnightly from June 2008 to June 2010. Water quality in the delta was generally good, despite high evapotranspiration rates which would normally produce very saline waters. Electrical conductivity and water temperature increased with distance from Mohembo to Lake Ngami, the former most likely due to evapoconcentration. In contrast, pH, DO, turbidity and TSS decreased with distance from Mohembo to Boro at the lower end of the seasonal floodplain, before increasing again to Lake Ngami. Dissolved oxygen and TSS most likely declined due to biological uptake and particle sedimentation, respectively. Strong and significant relationships were observed between TSS and turbidity and between DOC and EC, indicating that turbidity and EC could be useful proxies for routine estimations of TSS and DOC, respectively, in the delta.     

Macroinvertebrates as bioindicators of water quality in the Mkondoa River,Tanzania, in an agricultural area

The suitability of using macroinvertebrates as bioindicators of stream water quality was tested in the Mkondoa River in an agricultural area at Kilosa, using the rapid bioassessment protocol. The family biotic index (FBI) showed marked variation in water quality along the stream from values ranging from 4.1 to 5.0 in the upstream reaches, indicating good water quality, 5.3 to 5.5 in the mid-reaches and 6.0 to 6.5 in the lower reaches. The water quality index (WQI) indicated that water quality was fair (77 ± 0.98) in the upstream reach of the Mkondoa, marginal (55 ± 0.86) in the midstream reach and poor (33 ± 0.45) in the downstream reach. There were significant relationships between biological oxygen demand and dissolved oxygen and the occurrence of specific taxa, mainly Chironomus and Caenis. Significant changes in macroinvertebrate abundance were mostly related to changes in water quality. As in other parts of the world, macroinvertebrate communities proved to be good biological indicators of water quality and they should be used as bioindicators in long-term monitoring of this river.     

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

Synthesis of Free and Proliferating Cell Nuclear Antigen-bound Polyubiquitin Chains by the RING E3 Ubiquitin Ligase Rad5

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys⁶³-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13∼Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys⁶³ of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.DNA is susceptible to chemical alteration by many endogenous and exogenous agents. To counter this threat and maintain genome integrity, eukaryotic cells employ three main strategies: DNA repair pathways that directly reverse DNA damage, cell cycle checkpoints that allow time to repair the damage prior to replication, and DNA damage tolerance (DDT),² which is a method of bypassing DNA damage lesions during the DNA replication phase of the cell cycle.Proliferating cell nuclear antigen (PCNA) is a key regulatory protein in DNA replication and repair (1). At the replication fork, DNA is encircled by PCNA, a homotrimeric protein that promotes processive movement of the replicative DNA polymerase. Upon DNA damage and subsequent stalling of the replicative polymerase, Ub modifications of PCNA signal DDT, which allows a cell to bypass the lesion and proceed past this potential block in replication (2–4).In the DDT pathway, as in other Ub-dependent pathways, Ub is conjugated to a substrate by the actions of three enzymes, an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (5). The E1 enzyme initiates the pathway in a two-step reaction that utilizes ATP hydrolysis to activate the C terminus of Ub, culminating in the formation of an E1∼Ub thiolester. Subsequent transthiolation to the active site cysteine of the E2 generates an E2∼Ub thiolester. An E3 ligase then brings a substrate into close proximity to the E2∼Ub intermediate, thereby catalyzing the formation of an isopeptide bond between the amino group of a substrate lysine and the C-terminal glycine of Ub. Polyubiquitination occurs when this substrate is another Ub, either free or as part of a Ub-protein conjugate.The DDT pathway is characterized by distinct ubiquitination events on PCNA that occur in two stages (3, 4, 6). The first of these is monoubiquitination of lysine 164 on one or more of the PCNA subunits by the E2-E3 complex of Rad6-Rad18 in Saccharomyces cerevisiae (3, 4, 7). monoUb-PCNA can serve either as a signal for error-prone bypass of the DNA lesion by recruiting translesion polymerases or as a substrate for subsequent polyubiquitination by the E2 heterodimer Ubc13-Mms2 and the E3 ligase Rad5 (3, 4, 8, 9). The polyUb chain extended from the initial Ub moiety on monoUb-PCNA is linked specifically through Ub Lys⁶³ residues. This Lys⁶³-linked chain is thought to enable a template switch mechanism that allows for error-free bypass of the DNA lesion, in part by utilizing the single-strand DNA-dependent helicase activity of Rad5 (3, 4, 10, 11). Both PCNA ubiquitination events promote bypass of the DNA lesion rather than direct removal or repair of the lesion.We have been interested in the mechanism by which the yeast (Ubc13-Mms2)-Rad5 complex catalyzes the formation of Lys⁶³-linked polyUb on PCNA. Previous studies have shown that heterodimerization of the Ubc13-Mms2 E2 is essential for Lys⁶³-specific Ub-Ub conjugation in vitro and in vivo (12–15). Ubc13 is a canonical E2 enzyme with an active site cysteine that receives activated Ub by transthiolation from the E1∼Ub complex (12, 13). This Ub is referred to as the “donor Ub.” Mms2 is a Ub E2 variant protein that lacks the active site cysteine (12, 15); rather, Mms2 binds to a second Ub, the “acceptor Ub,” and positions it to facilitate nucleophilic attack on the Ubc13∼Ub thiolester bond by the ϵ-amine of Lys⁶³ (15, 16). The positioning of the acceptor Ub by Mms2 controls the specificity of polyUb assembly such that only Lys⁶³-linked chains can be formed (16).Ubc13-Mms2 can synthesize Lys⁶³-linked chains in vitro in the absence of a PCNA substrate or an E3 ligase (12, 13). However, unlike the synthesis of free Lys⁶³-linked polyUb chains by Ubc13-Mms2, little is known about the polyubiquitination of PCNA or the role of the Rad5 E3 ligase in these reactions. Rad5 can bind PCNA and Rad18, and it contains a catalytic RING domain that characterizes the largest class of E3 ligases (17–21). There is evidence that RING E3s like Rad5 may play a more active role in ubiquitination than simply bringing the substrate into close proximity with the E2∼Ub. Several RING E3s have been shown to stimulate the synthesis of unanchored polyUb chains or autoubiquitination of their cognate E2s in the absence of substrates (22–24). This stimulation may be related to the ability of RING E3s to enhance reactivity of the E2∼Ub thiolester bond through allosteric effects (25, 26).Using purified recombinant forms of Ubc13, Mms2, and Rad5, we have explored the assembly of free Lys⁶³-linked polyUb chains as well as the extension of a polyUb chain on a synthetic analog of monoUb-PCNA. We show that Rad5 facilitates ubiquitination in part by increasing the reactivity of the Ubc13∼Ub thiolester bond. With monoUb-PCNA substrates, Rad5 also stimulated polyubiquitination through direct interactions with PCNA and recruitment of Ub-charged Ubc13-Mms2. Surprisingly, Rad5 recognition of monoUb-PCNA appeared to depend on interactions only with the PCNA moiety of the conjugate, which suggests that substrate selectivity in vivo is likely to depend on additional factors.     

Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion

Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

An improved SUMmOn‐based methodology for the identification of ubiquitin and ubiquitin‐like protein conjugation sites identifies novel ubiquitin‐like protein chain linkages

Ubiquitin (Ub) and the ubiquitin‐like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half‐life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis‐database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine‐rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin‐related modifier (SUMO) chain (SUMO‐2 K42, SUMO‐3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.