PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.     

Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder

We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.     

An enzymatic atavist revealed in dual pathways for water activation

Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the biosynthesis of guanine nucleotides. This reaction involves two different chemical transformations, an NAD-linked redox reaction and a hydrolase reaction, that utilize mutually exclusive protein conformations with distinct catalytic residues. How did Nature construct such a complicated catalyst? Here we employ a “Wang-Landau” metadynamics algorithm in hybrid quantum mechanical/molecular mechanical (QM/MM) simulations to investigate the mechanism of the hydrolase reaction. These simulations show that the lowest energy pathway utilizes Arg418 as the base that activates water, in remarkable agreement with previous experiments. Surprisingly, the simulations also reveal a second pathway for water activation involving a proton relay from Thr321 to Glu431. The energy barrier for the Thr321 pathway is similar to the barrier observed experimentally when Arg418 is removed by mutation. The Thr321 pathway dominates at low pH when Arg418 is protonated, which predicts that the substitution of Glu431 with Gln will shift the pH-rate profile to the right. This prediction is confirmed in subsequent experiments. Phylogenetic analysis suggests that the Thr321 pathway was present in the ancestral enzyme, but was lost when the eukaryotic lineage diverged. We propose that the primordial IMPDH utilized the Thr321 pathway exclusively, and that this mechanism became obsolete when the more sophisticated catalytic machinery of the Arg418 pathway was installed. Thus, our simulations provide an unanticipated window into the evolution of a complex enzyme.     

A genomic analysis of the archaeal system Ignicoccus hospitalis-Nanoarchaeum equitans

Background

The relationship between the hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans is the only known example of a specific association between two species of Archaea. Little is known about the mechanisms that enable this relationship.

Results

We sequenced the complete genome of I. hospitalis and found it to be the smallest among independent, free-living organisms. A comparative genomic reconstruction suggests that the I. hospitalis lineage has lost most of the genes associated with a heterotrophic metabolism that is characteristic of most of the Crenarchaeota. A streamlined genome is also suggested by a low frequency of paralogs and fragmentation of many operons. However, this process appears to be partially balanced by lateral gene transfer from archaeal and bacterial sources.

Conclusions

A combination of genomic and cellular features suggests highly efficient adaptation to the low energy yield of sulfur-hydrogen respiration and efficient inorganic carbon and nitrogen assimilation. Evidence of lateral gene exchange between N. equitans and I. hospitalis indicates that the relationship has impacted both genomes. This association is the simplest symbiotic system known to date and a unique model for studying mechanisms of interspecific relationships at the genomic and metabolic levels.     

Substrate water binding and oxidation in photosystem II

This mini review presents a general introduction to photosystem II with an emphasis on the oxygen evolving complex. An attempt is made to summarise what is currently known about substrate interaction in the oxygen evolving complex of photosystem II in terms of the nature of the substrate, the timing and the location of its binding. As the nature of substrate water binding has a direct bearing on the mechanism of O-O bond formation in PSII, a discussion of O-O bond formation follows the summary of current opinion in substrate interaction.     

Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions (review)

Integrins are heterodimeric membrane-spanning adhesion receptors that are essential for a wide range of biological functions. Control of integrin conformational states is required for bidirectional signalling across the membrane. Key components of this control mechanism are the transmembrane and cytoplasmic domains of the alpha and beta subunits. These domains are believed to interact, holding the integrin in the inactive state, while inside-out integrin activation is accompanied by domain separation. Although there are strong indications for domain interactions, the majority of evidence is insufficient to precisely define the interaction interface. The current best model of the complex, derived from computational calculations with experimental restraints, suggests that integrin activation by the cytoplasmic protein talin is accomplished by steric disruption of the alpha/beta interface. Better atomic-level resolution structures of the alpha/beta transmembrane/cytoplasmic domain complex are still required for the resting state integrin to corroborate this. Integrin activation is also controlled by competitive interactions involving the cytoplasmic domains, particularly the beta-tails. The concept of the beta integrin tail as a focal adhesion interaction 'hub' for interactions and regulation is discussed. Current efforts to define the structure and affinity of the various complexes formed by integrin tails, and how these interactions are controlled, e.g. by phosphorylation and localization, are described.     

Healing by primary closure versus open healing after surgery for pilonidal sinus: systematic review and meta-analysis

Objective To determine the relative effects of open healing compared with primary closure for pilonidal sinus and optimal closure method (midline v off-midline).Design Systematic review and meta-analyses of randomised controlled trials.Data sources Cochrane register of controlled trials, Cochrane Wounds Group specialised trials register, Medline (1950-2007), Embase, and CINAHL bibliographic databases, without language restrictions.Data extraction Primary outcomes were time (days) to healing, surgical site infection, and recurrence rate. Secondary outcomes were time to return to work, other complications and morbidity, cost, length of hospital stay, and wound healing rate.Study selection Randomised controlled trials evaluating surgical treatment of pilonidal sinus in patients aged 14 years or more. Data were extracted independently by two reviewers and assessed for quality. Meta-analyses used fixed and random effects models, dichotomous data were reported as relative risks or Peto odds ratios and continuous data are given as mean differences; all with 95% confidence intervals.Results 18 trials (n=1573) were included. 12 trials compared open healing with primary closure. Time to healing was quicker after primary closure although data were unsuitable for aggregation. Rates of surgical site infection did not differ; recurrence was less likely to occur after open healing (relative risk 0.42, 0.26 to 0.66). 14 patients would require their wound to heal by open healing to prevent one recurrence. Six trials compared surgical closure methods (midline v off-midline). Wounds took longer to heal after midline closure than after off-midline closure (mean difference 5.4 days, 95% confidence interval 2.3 to 8.5), rate of infection was higher (relative risk 4.70, 95% confidence interval 1.93 to 11.45), and risk of recurrence higher (Peto odds ratio 4.95, 95% confidence interval 2.18 to 11.24). Nine patients would need to be treated by an off-midline procedure to prevent one surgical site infection and 11 would need to be treated to prevent one recurrence.Conclusions Wounds heal more quickly after primary closure than after open healing but at the expense of increased risk of recurrence. Benefits were clearly shown with off-midline closure compared with midline closure. Off-midline closure should become standard management for pilonidal sinus when closure is the desired surgical option.