Inherent asymmetry in the 26S proteasome is defined by the ubiquitin receptor RPN13.

The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps.

Criteria for reflex peripheral smear review in infants.

Criteria for peripheral smear review are designed to include those samples with results outside the reference interval and can be more extreme based on what is considered to have clinical utility. However, we are unaware of previous studies that reported the distributions of various complete blood cell count (CBC) parameters in infants. In the following study we reviewed screening CBC results of 692 infants aged 9-15 months in order to determine the proportion of peripheral smear reviews recommended according to consensus criteria and that after adjusting for the observed distributions of the various parameters. According to consensus criteria the recommended reflex peripheral smear review rate was 39.7% (95% CI 36.1-43.4) whereas after adjustment for the observed distributions, the rate fell to 5.6% (95% CI 3.9-7.3) (p < 0.001). The major reasons for the difference in rates were the high proportion of infants with an absolute lymphocyte count > 7 × 10(9)/L (17.5%), the presence of a plus one blast flag (4.3%), and a large unstained cell count of ≥ 5% (26.2%) (equivalent to + 1 atypical flag). We found that international consensus criteria for reflex peripheral smear review results in a very high peripheral smear review rate in well infants, and might be inappropriate.

Stalled proteasomes are directly relieved by P97 recruitment.

The 26 S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation, these include regulation of substrate delivery, deubiquitination, unfolding, and 20 S gate dilation. Here we show a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26 S proteasome via the 19 S particle thereby forming an additional hexameric ATPase ring to relieve repression. P97-bound proteasomes showed selective binding toward the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97-bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new checkpoint in UPS impairment. The ability to modulate proteasome activity and properly respond to protein misfolding, is of great importance in cellular regulation.

Trichostatin A regulates peroxiredoxin expression and virulence of the parasite Entamoeba histolytica.

Histone deacetylation is associated with a repressed chromatin state, and histone acetylase and deacetylase activities have been previously described in Entamoeba histolytica. To investigate their roles in the control of Entamoeba gene expression, the parasite was grown in 50 nM trichostatin A (TSA), an inhibitor of histone deacetylase. TSA enhanced the cytopathic and hemolytic activity of the parasite and its resistance to oxidative stress. We first focused our attention on peroxiredoxin, a protein previously associated with E. histolytica virulence and resistance to oxidative stress. We found that the expression of peroxiredoxin was increased after TSA treatment, but were unable to confirm that this was a direct consequence of histone modification at the promoter. By microarray analysis, we found that some other mRNAs encoding some other virulence factors, such as the galactose-inhibitable lectin small subunits, were also increased. The pattern of gene expression was surprisingly different from that previously described after treatment with 150 nM TSA.

Anemia and estimated glomerular filtration rates.

OBJECTIVE: To determine the relationship between the estimated glomerular filtration rate (eGFR) and the prevalence of anemia that has potential implications for reporting results of the eGFR. METHODS: Serum creatinine and hemoglobin test results from 18,474 outpatients aged 50 years or older were reviewed. We calculated the eGFR using the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI) and the Modification of Diet in Renal Disease equation (MDRD) and determined the odds of anemia (according to the World Health Organization definition) at various eGFR levels, adjusted for age and gender. RESULTS: The lowest proportion of anemia was observed in those with an eGFR of 80-89 mL/min per 1.73 m(2) and 90-99 mL/min per 1.73 m(2) (MDRD and CKD-EPI respectively), with an increasing prevalence of anemia in those with either an eGFR of 60-69 mL/min per 1.73 m(2) or 100-109 mL/min per 1.73 m(2) calculated by either equation (p<0.05) with a dose-response effect. CONCLUSIONS: We found a U-shaped relationship between anemia and the eGFR, suggesting that values >60 mL per 1.73 m(2) should be reported. However, the clinical utility and potential side effects of reporting such values need to be determined. Also, these preliminary findings require confirmation by studies in other settings.

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome.

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.

Sensing DNA methylation in the protozoan parasite Entamoeba histolytica.

In the protozoan parasite Entamoeba histolytica, 5-methylcytosine (m5C) was found predominantly in repetitive elements. Its formation is catalysed by Ehmeth, a DNA methyltransferase that belongs to the Dnmt2 subfamily. Here we describe a 32 kDa nuclear protein that binds in vitro with higher affinity to the methylated form of a DNA encoding a reverse transcriptase of an autonomous non-long-terminal repeat retrotransposon (RT LINE) compared with the non-methylated RT LINE. This protein, named E. histolytica-methylated LINE binding protein (EhMLBP), was purified from E. histolytica nuclear lysate, identified by mass spectrometry, and its corresponding gene was cloned. EhMLBP corresponds to a gene of unknown function that shares strong homology with putative proteins present in Entamoeba dispar and Entamoeba invadens. In contrast, the homology dropped dramatically when non-Entamoebidae sequences were considered and only a weak sequence identity was found with Trypanosoma and several prokaryotic histone H1. Recombinant EhMLBP showed the same binding preference for methylated RT LINE as the endogenous EhMLBP. Deletion mapping analysis localized the DNA binding region at the C-terminal part of the protein. This region is sufficient to assure the binding to methylated RT LINE with high affinity. Western blot and immunofluorescence microscopy, using an antibody raised against EhMLBP, showed that it has a nuclear localization. Chromatin immunoprecipitation (ChIP) confirmed that EhMLBP interacts with RT LINE in vivo. Finally, we showed that EhMLBP can also bind rDNA episome, a DNA that is methylated in the parasite. This suggests that EhMLBP may serve as a sensor of methylated repetitive DNA. This is the first report of a DNA-methylated binding activity in protozoa.