Use of detergents to study membrane rafts: the good, the bad, and the ugly

Eukaryotic cell membranes contain microdomains called lipid rafts, which are cholesterol-rich domains in which lipid acyl chains are tightly packed and highly extended. A variety of proteins associate preferentially with rafts, and this raft association is important in a wide range of functions. A powerful and widely-used method for studying lipid rafts takes advantage of their insolubility in non-ionic detergents. Here we describe the basis of detergent insolubility, and review strengths, limitations, and unresolved puzzles regarding this method.     

Critical contribution of the MDM2 acidic domain to p53 ubiquitination

MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.     

Molecular basis of p53 functional inactivation by the leukemic protein MLL-ELL

The Eleven Lysine-rich Leukemia (ELL) gene undergoes translocation and fuses in frame to the Multiple Lineage Leukemia (MLL) gene in a substantial proportion of patients suffering from acute forms of leukemia. Molecular mechanisms of cellular transformation by the MLL-ELL fusion are not well understood. Although both MLL-ELL and wild-type ELL can reduce functional activity of p53 tumor suppressor, our data reveal that MLL-ELL is a much more efficient inhibitor of p53 than is wild-type ELL. We also demonstrate for the first time that ELL extreme C terminus [ELL(eCT)] is required for the recruitment of p53 into MLL-ELL nuclear foci and is both necessary and sufficient for the MLL-ELL inhibition of p53-mediated induction of p21 and apoptosis. Finally, our results demonstrate that MLL-ELL requires the presence of intact ELL(eCT) in order to disrupt p53 interactions with p300/CBP coactivator and thus significantly reduce p53 acetylation in vivo. Since ELL(eCT) has recently been shown to be both necessary and sufficient for MLL-ELL-mediated transformation of normal blood progenitors, our data correlate ELL(eCT) contribution to MLL-ELL transformative effects with its ability to functionally inhibit p53.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Sex Differences in the Default Mode Network with Regard to Autism Spectrum Traits: A Resting State fMRI Study

Autism spectrum traits exist on a continuum and are more common in males than in females, but the basis for this sex difference is unclear. To this end, the present study draws on the extreme male brain theory, investigating the relationship between sex difference and the default mode network (DMN), both known to be associated with autism spectrum traits. Resting-state functional magnetic resonance imaging (MRI) was carried out in 42 females (mean age ± standard deviation, 22.4 ± 4.2 years) and 43 males (mean age ± standard deviation, 23.8 ± 3.9 years) with typical development. Using a combination of different analyses (viz., independent component analysis (ICA), fractional amplitude of low-frequency fluctuation (fALFF), regional homogeneity (ReHo), and seed-based analyses), we examined sex differences in the DMN and the relationship to autism spectrum traits as measured by autism-spectrum quotient (AQ) scores. We found significant differences between female and male subjects in DMN brain regions, with seed-based analysis revealing a significant negative correlation between default-mode resting state functional connectivity of the anterior medial prefrontal cortex seed (aMPFC) and AQ scores in males. However, there were no relationships between DMN sex differences and autism spectrum traits in females. Our findings may provide important insight into the skewed balance of functional connectivity in males compared to females that could serve as a potential biomarker of the degree of autism spectrum traits in line with the extreme male brain theory.     

Insular Gray Matter Volume and Objective Quality of Life in Schizophrenia

Improving quality of life has been recognized as an important outcome for schizophrenia treatment, although the fundamental determinants are not well understood. In this study, we investigated the association between brain structural abnormalities and objective quality of life in schizophrenia patients. Thirty-three schizophrenia patients and 42 age-, sex-, and education-matched healthy participants underwent magnetic resonance imaging. The Quality of Life Scale was used to measure objective quality of life in schizophrenia patients. Voxel-based morphometry was performed to identify regional brain alterations that correlate with Quality of Life Scale score in the patient group. Schizophrenia patients showed gray matter reductions in the frontal, temporal, limbic, and subcortical regions. We then performed voxel-based multiple regression analysis in these regions to identify any correlations between regional gray matter volume and Quality of Life Scale scores. We found that among four subcategories of the scale, the Instrumental Role category score correlated with gray matter volume in the right anterior insula in schizophrenia patients. In addition, this correlation was shown to be mediated by negative symptoms. Our findings suggest that the neural basis of objective quality of life might differ topographically from that of subjective QOL in schizophrenia.     

Syntheses of water-soluble [60]fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells

Water-soluble [60]fullerene (C₆₀) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C₆₀ derivatives together with some reported ones were tested. Among the compounds tested, C₆₀/(γ-CyD)₂, C₆₀-bis(γ-CyD) (5) containing C₆₀-mono(γ-CyD) (5′), and C₆₀/PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.     

Novel membrane-localizing TEMPO derivatives for measurement of cellular oxidative stress at the cell membrane

Oxidative stress affecting lipid membranes is considered to be closely related to cardiovascular disease and brain ischemia. In this study, we designed and synthesized membrane-localizing TEMPO derivatives and demonstrated that one of these synthesized probes, compound 1, localized and detected oxidative stress in the cell membrane in an endotoxic model of a mouse macrophage-like cell line. Compound 1 is therefore a potentially useful probe for evaluating oxidative stress at the cell membrane.