The Laforin–Malin Complex,Involved in Lafora Disease,Promotes the Incorporation of K63-linked Ubiquitin Chains into AMP-activated Protein Kinase β Subunits

Lafora progressive myoclonus epilepsy is a fatal neurodegenerative disorder caused by defects in the function of at least two proteins: laforin, a dual-specificity protein phosphatase, and malin, an E3-ubiquitin ligase. In this study, we report that a functional laforin–malin complex promotes the ubiquitination of AMP-activated protein kinase (AMPK), a serine/threonine protein kinase that acts as a sensor of cellular energy status. This reaction occurs when any of the three AMPK subunits (α, β, and γ) are expressed individually in the cell, and it also occurs on AMPKβ when it is part of a heterotrimeric complex. We also report that the laforin–malin complex promotes the formation of K63-linked ubiquitin chains, which are not involved in proteasome degradation. On the contrary, this modification increases the steady-state levels of at least AMPKβ subunit, possibly because it leads to the accumulation of this protein into inclusion bodies. These results suggest that the modification introduced by the laforin–malin complex could affect the subcellular distribution of AMPKβ subunits.     

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 <Emphasis Type="Italic">in vitro</Emphasis>

Introduction  

Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.     

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

Background  

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear.     

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls

Background

Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation.

Methods

Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, β-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n?=?37) were compared with levels in non-FAP patient controls (n?=?16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n?=?14) or celecoxib & placebo (n?=?13) were evaluated in patients with FAP.

Results

mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p?=?0.008) and caspase-3 (3.30% vs. 5.31%, p?=?0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA.

Conclusions

Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa.

Trial registration

http://ClinicalTrials.gov number NCT00808743

     

Mitochondrial mismatch analysis is insensitive to the mutational process

Mismatch distributions are histograms showing the pattern of nucleotide (or restriction) site differences between pairs of individuals in a sample. They can be used to test hypotheses about the history of population size and subdivision (if selective neutrality is assumed) or about selection (if a constant population size is assumed). Previous work has assumed that mutations never strike the same site twice, an assumption that is called the model of infinite sites. Fortunately, the results are surprisingly robust even when this assumption is violated. We show here that (1) confidence regions inferred using the infinite- sites model differ little from those inferred using a model of finite sites with uniform site-specific mutation rates, and (2) even when site- specific mutation rates follow a gamma distribution, confidence regions are little changed until the gamma shape parameter falls well below its plausible range, to roughly 0.01. In addition, we evaluate and reject the proposition that mismatch waves are produced by pooling data from several subdivisions of a structured population.      

Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a "parental" peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.