Identification of proteins potentially involved in the formation of Lafora bodies, a hallmark of Lafora disease

Lafora Disease (LD) is a fatal teenage-onset progressive myoclonus epilepsy. It is characterized by the formation of Lafora bodies (LBs), deposits of abnormally branched, insoluble, hyperphosphorylated glycogen-like polymers that are generally believed to trigger the development of the clinical symptoms of LD. 58% and 35% of the LD cases are caused by mutations in EPM2A (laforin) and EPM2B (malin), respectively. However, little is known about their function in LB formation. Two different mechanisms have been proposed to explain the accumulation of insoluble LBs: first, excessive glycogen phosphorylation and, second, an imbalance between glycogen synthesizing enzymes. The present study aims at the identification of proteins involved in the molecular mechanisms leading to LB formation and appearance of LD and the phosphorylation of glycogen

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

BACKGROUND: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. PRINCIPAL FINDINGS: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. CONCLUSIONS: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades

Transcriptome Analysis in Tardigrade Species Reveals Specific Molecular Pathways for Stress Adaptations

Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant

Investigating the Proteome of Tardigrades: Towards a Better Understanding of Molecular Mechanisms during Anhydrobiosis

Tardigrades have fascinated researchers for more than 300 years because of their amazing capability to undergo anhydrobiosis. In extreme states of dehydration, anhydrobiotic tardigrades undergo a metabolic dormancy, in which metabolism decreases to a non-measurable level and life comes to a reversible standstill until activity is resumed under more favourable conditions. In the anhydrobiotic (tun) state, tardigrades are extraordinary tolerant to physical extremes including high and subzero temperatures, high pressure, and extreme levels of ionizing radiation. Possessing the ability to enter this ametabolic state at any developmental stage, tardigrades are capable of surviving for a very long time and extend their lifespan significantly. Anhydrobiosis seems to be the result of dynamic processes and appears to be mediated by protective systems that prevent lethal damage. However, the survival mechanisms of tardigrades are still poorly understood. This is mainly caused by notable absence of detailed analysis concerning the proteome and genome of these organisms. FUNCRYPTA (Functional Analysis of Dynamic Processes in Cryptobiotic Tardigrades) project consisting of four research groups has been established to fill this gap by performing a broad range of investigations and analyses. As Funcrypta´s cooperation partner specialized in proteomics field we started with establishing optimal protocols for extraction of proteins from tardigrades, performing high resolution gel electrophoresis and high throughput protein identification and quantification. Since the presence of a comprehensive protein database is a prerequisite for protein identification, a M. tardigradum sequencing project has been initiated in parallel to our proteomic study by our genomic cooperation partner. The first tardigrade protein database translated from expressed sequence tags (ESTs), that have been generated by Sanger sequencing contained 3318 sequences. This protein database allowed us to develop the first proteome map of tardigrades utilizing 2D gel electrophoresis. The second protein database based on 454 sequencing with a high number of 24679 protein sequences provided us the basis for protein identification and quantification in a large scale. This resulted for the first time in a broad characterization of proteins expressed in tardigrades. More than 3000 unique proteins of M. tardigradum in three different states (early embryonic state and adults in active and anhydrobiotic states) have been identified with high sequence coverage using 1D electrophoresis in combination with high sensitive nanoLC ESI-MS/MS on a LTQ-Orbitrap mass spectrometer. Among the broad range of identified protein families, proteins known to be associated with desiccation tolerance were identified. This includes proteins with antioxidant activity, chaperones in particular heat shock proteins, aquaporins and Late Embryogenesis Abundant (LEA) proteins. Furthermore the present study provides a semi-quantitative analysis of proteins expressed in early embryonic state and adults in active and anhydrobiotic states using a label-free approach based on Exponentially Modified Protein Abundance Index (emPAI). This method allowed the classification of proteins present in one state in major and minor components and furthermore a quantitative analysis of differentially expressed proteins in each state. The semi-quantitative analysis delivered consequential results in comparing early embryonic state and adults, which will be of importance in the field of developmental biology. Using this approach we quantitatively analyzed the expressed heat shock proteins in active and tun states. The success of the analysis could be confirmed, by the published gene expression analysis of some heat shock proteins performed by our cooperation partner, which delivered similar results. The semi-quantitative analysis of active versus tun state demonstrated up-regulation of proteins in tun state that are mainly not annotated, since they are tardigrade specific and the homology search delivered no result. The functional analysis of these specific proteins in future investigations will be of major importance in regard to investigating anhydrobiosis. Analyzing the proteins that are only identified in tun state, leads to the assumption that not only proteins such as chaperones play important roles in protection mechanisms during anhydrobiosis, but also further processes and mechanisms are associated such as phosphorylation and activation of intracellular signalling cascades. Therefore optimal protocols for analyzing phosphoproteins in tardigrades have been developed and first experiments in detecting phosphoproteins on 2D gels using fluorescent dye (ProQ Diamond) have been performed. This comprehensive study from the first step of developing optimized protocol for protein extraction to the large scale protein identification and quantification builds the basis for future investigations in the field of anhydrobiotic organisms in regard to isolation and functional characterization of proteins, which are associated with protection mechanisms during anhydrobiosis. Understanding the desiccation-tolerance in anhydrobiotic tardigrades will probably enable us to develop new strategies for long-term stabilization and preservation of biological macromolecules in the future, which will be immensely important in medical field as well as in pharmaceutical industry

Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state.

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state

Comparative proteome analysis of proteins identified in different states.

<p>The Venn diagram illustrates the number of protein identifications in EES, AS and TS. A total of 1301 proteins were found in all three states, 472 proteins are found only in EES (a) and 680 proteins are found only in adult tardigrades in TS and AS (f). Proteins which are non-overlapping (a, b, c) or partially overlapping (d, e, f) between the different states are analyzed using Blast2GO program to determine the involved biological processes. The ten major biological processes for non-overlapping proteins are listed in 2a-2c and for partially overlapping proteins in 2d–2f.</p

Major protein components in adult tardigrades in active and tun state. 53 proteins were found as major components in adult tardigrades in AS and 49 in TS.

<p>Comparing the annotated proteins in AS and TS we found the same two major functional groups, protein members of structural constituent of cytoskeleton/muscle and protein members of large lipid transporter family. The contig description is indicated with asterisk, in case we found putative candidates in DomainSweep analysis.</p