Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. Methodology/Principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. Conclusions/Significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovación (MICINN) to A

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR

Depletion of AADC activity in caudate nucleus and putamen of Parkinson’s disease patients; implications for ongoing AAV2-AADC gene therapy trial

In Parkinson's disease (PD), aromatic L-amino acid decarboxylase (AADC) is the rate-limiting enzyme in the conversion of L-DOPA (Sinemet) to dopamine (DA). Previous studies in PD animal models demonstrated that lesion of dopaminergic neurons is associated with profound loss of AADC activity in the striatum, blocking efficient conversion of L-DOPA to DA. Relatively few studies have directly analyzed AADC in PD brains. Thus, the aim of this study was to gain a better understanding of regional changes in AADC activity, DA, serotonin and their monoamine metabolites in the striatum of PD patients and experimentally lesioned animals (rat and MPTP-treated nonhuman primate, NHP). Striatal AADC activity was determined post mortem in neuropathologically confirmed PD subjects, animal models and controls. A regional analysis was performed for striatal AADC activity and monoamine levels in NHP tissue. Interestingly, analysis of putaminal AADC activity revealed that control human striatum contained much less AADC activity than rat and NHP striata. Moreover, a dramatic loss of AADC activity in PD striatum compared to controls was detected. In MPTP-treated NHP, caudate nucleus was almost as greatly affected as putamen, although mean DA turnover was higher in caudate nucleus. Similarly, DA and DA metabolites were dramatically reduced in different regions of PD brains, including caudate nucleus, whereas serotonin was relatively spared. After L-DOPA administration in MPTP-treated NHP, very poor conversion to DA was detected, suggesting that AADC in NHP nigrostriatal fibers is mainly responsible for L-DOPA to DA conversion. These data support further the rationale behind viral gene therapy with AAV2-hAADC to restore AADC levels in putamen and suggest further the advisability of expanding vector delivery to include coverage of anterior putamen and the caudate nucleus

Gene Therapy for Misfolding Protein Diseases of the Central Nervous System

Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. Accordingly, most recent studies aim to prevent protein misfolding and/or aggregation as a strategy to treat these pathologies. For instance, state-of-the-art approaches, such as silencing protein overexpression by means of RNA interference, are being tested with positive outcomes in preclinical models of animals overexpressing the corresponding protein. Therapies designed to treat central nervous system diseases should provide accurate delivery of the therapeutic agent and long-term or chronic expression by means of a nontoxic delivery vehicle. After several years of technical advances and optimization, gene therapy emerges as a promising approach able to fulfill those requirements. In this review we will summarize the latest improvements achieved in gene therapy for central nervous system diseases associated with protein misfolding (e.g., amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases), as well as the most recent approaches in this field to treat these pathologies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13311-013-0191-8) contains supplementary material, which is available to authorized users