Phosphorylation of Serine 51 Regulates the Interaction of Human DNA Ligase I with Replication Factor C and Its Participation in DNA Replication and Repair

Human DNA ligase I (hLigI) joins Okazaki fragments during DNA replication and completes excision repair via interactions with proliferating cell nuclear antigen and replication factor C (RFC). Unlike proliferating cell nuclear antigen, the interaction with RFC is regulated by hLigI phosphorylation. To identity of the site(s) involved in this regulation, we analyzed phosphorylated hLigI purified from insect cells by mass spectrometry. These results suggested that serine 51 phosphorylation negatively regulates the interaction with RFC. Therefore, we constructed versions of hLigI in which serine 51 was replaced with either alanine (hLigI51A) to prevent phosphorylation or aspartic acid (hLigI51D) to mimic phosphorylation. hLigI51D but not hLigI51A was defective in binding to purified RFC and in associating with RFC in cell extracts. Although DNA synthesis and proliferation of hLigI-deficient cells expressing either hLig51A or hLig51 was reduced compared with cells expressing wild-type hLigI, cellular senescence was only observed in the cells expressing hLigI51D. Notably, these cells had increased levels of spontaneous DNA damage and phosphorylated CHK2. In addition, although expression of hLigI51A complemented the sensitivity of hLigI-deficient cells to a poly (ADP-ribose polymerase (PARP) inhibitor, expression of hLig151D did not, presumably because these cells are more dependent upon PARP-dependent repair pathways to repair the damage resulting from the abnormal DNA replication. Finally, neither expression of hLigI51D nor hLigI51A fully complemented the sensitivity of hLigI-deficient cells to DNA alkylation. Thus, phosphorylation of serine 51 on hLigI plays a critical role in regulating the interaction between hLigI and RFC, which is required for efficient DNA replication and repair.     

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

Background

Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes.

Case presentation

All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced.

Conclusion

A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission.     

Burkholderia cepacia complex infection in a cohort of Italian patients with cystic fibrosis

The aims of this study were to detect Burkholderia cepacia complex (Bcc) strains in a cohort of Cystic Fibrosis patients (n=276) and to characterize Bcc isolates by molecular techniques. The results showed that 11.23% of patients were infected by Bcc. Burkholderia cenocepacia lineage III-A was the most prevalent species (64.3%) and, of these, 10% was cblA positive and 50% esmR positive. Less than half of the strains were sensitive to ceftazidime, meropenem, piperacillin tazobactam, and trimethoprim-sulfamethoxazole. About half of the strains (41%) had homogeneous profiles, suggesting cross-transmission. The infection by B. cenocepacia was associated to a high rate of mortality (p=0.01).     

Application of quantitative proteomics to the integrated analysis of the ubiquitylated and global proteomes of xenograft tumor tissues

Background

Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues.

Results

In this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level.

Conclusions

This is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9086-5) contains supplementary material, which is available to authorized users.     

Soil nutrients under shrub hummocks and debris mounds two decades after ploughing

Aims

Shrub removal by ploughing has been used widely to reduce the effects of shrub encroachment into open woodlands and grasslands. Our aim was to demonstrate that soil chemical properties varied markedly among three patch types (shrub hummock, debris mound, interspace) which varied in age, almost two decades after shrub removal by ploughing.

Methods

We compared changes in nutrients under 1) young post-ploughing recruits and mature, unploughed shrubs, 2) mature and recently formed debris mounds and 3) ploughed and recovering interspaces at three depths.

Results

Irrespective of their age, nutrient concentrations were greater under shrub hummocks and debris mounds than in the interspaces at two sites. Soil in mature shrub hummocks generally had greater levels of labile carbon and nitrogen (total, mineral, mineralisable), but results varied between sites. There were a few, sometimes inconsistent, effects of ploughing on nutrients under debris mounds, and no differences between the interspaces two decades after ploughing. Nutrient effects were most marked in the top 15 cm of the soil, diminishing rapidly with depth.

Conclusions

Our results reinforce the importance of hummocks and mounds as resource sinks and indicate the long-lasting effects of disturbances such as ploughing on soil nutrient pools.