A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity.

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Correction: Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

[This corrects the article DOI: 10.1371/journal.pbio.3000414.].

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.

Revealing the Venomous Secrets of the Spider's Web.

Orb-weaving spiders use a highly strong, sticky and elastic web to catch their prey. These web properties alone would be enough for the entrapment of prey; however, these spiders may be hiding venomous secrets in the web, which current research is revealing. Here, we provide strong proteotranscriptomic evidence for the presence of toxin/neurotoxin-like proteins, defensins, and proteolytic enzymes on the web silk from Nephila clavipes spider. The results from quantitative-based transcriptomic and proteomic approaches showed that silk-producing glands produce an extensive repertoire of toxin/neurotoxin-like proteins, similar to those already reported in spider venoms. Meanwhile, the insect toxicity results demonstrated that these toxic components can be lethal and/or paralytic chemical weapons used for prey capture on the web, and the presence of fatty acids in the web may be a responsible mechanism opening the way to the web toxins for accessing the interior of prey's body, as shown here. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among two spider groups, Araneomorphae and Mygalomorphae, and the findings showed protein sequences similar to toxins found in the taxa Scorpiones and Hymenoptera in addition to Araneae. Overall, these data represent a valuable resource to further investigate other spider web toxin systems and also suggest that N. clavipes web is not a passive mechanical trap for prey capture, but it exerts an active role in prey paralysis/killing using a series of neurotoxins.

Proteome Changes Paralleling the Olfactory Conditioning in the Forager Honey Bee and Provision of a Brain Proteomics Dataset.

The olfactory conditioning of the bee proboscis extension reflex (PER) is extensively used as a paradigm in associative learning of invertebrates but with limited molecular investigations. To investigate which protein changes are linked to olfactory conditioning, a non-sophisticated conditioning model is applied using the PER in the honeybee (Apis mellifera). Foraging honeybees are assigned into three groups based on the reflex behavior and training: conditioned using 2-octanone (PER-conditioned), and sucrose and water controls. Thereafter, the brain synaptosomal proteins are isolated and analyzed by quantitative proteomics using stable isotope labeling (TMT). Additionally, the complex proteome dataset of the bee brain is generated with a total number of 5411 protein groups, including key players in neurotransmitter signaling. The most significant categories affected during olfactory conditioning are associated with "SNARE interactions in vesicular transport" (BET1 and VAMP7), ABC transporters, and fatty acid degradation pathways.

Spheroid glioblastoma culture conditions as antigen source for dendritic cell-based immunotherapy: spheroid proteins are survival-relevant targets but can impair immunogenic interferon γ production.

BACKGROUND: Glioblastoma is the most aggressive type of brain cancer. Dendritic cell (DC)-based immunotherapy against glioblastoma depends on the effectiveness of loaded antigens. Sphere-inducing culture conditions are being studied by many as a potential antigen source. Here, we investigated two different in vitro conditions (spheroid culture versus adherent culture) in relation to DC immunotherapy: (1) We studied the specific spheroid-culture proteome and assessed the clinical importance of spheroid proteins. (2) We evaluated the immunogenicity of spheroid lysate - both compared to adherent conditions. METHODS: We used seven spheroid culture systems, three of them patient-derived. Stemness-related markers were studied in those three via immunofluorescence. Spheroid-specific protein expression was measured via quantitative proteomics. The Cancer Genome Atlas (TCGA) survival data was used to investigate the clinical impact of spheroid proteins. Immunogenicity of spheroid versus adherent cell lysate was explored in autologous ELISPOT systems (DCs and T cells from the three patients). RESULTS: (1) The differential proteome of spheroid versus adherent glioblastoma culture conditions could successfully be established. The top 10 identified spheroid-specific proteins were associated with significantly decreased overall survival (TCGA MIT/Harvard cohort; n = 350, P = 0.014). (2) In exploratory experiments, immunogenicity of spheroid lysate vis-á-vis interferon (IFN)γ production was lower than that of adherent cell lysate (IFNγ ELISPOT; P = 0.034). CONCLUSIONS: Spheroid culture proteins seem to represent survival-relevant targets, supporting the use of spheroid culture conditions as an antigen source for DC immunotherapy. However, immunogenicity enhancement should be considered for future research. Transferability of our findings in terms of clinical impact and regarding different spheroid-generation techniques needs further validation.

Proteomic Studies on the Swim Bladder of the European Eel (Anguilla anguilla).

The swim bladder of a fish is a vital organ that with gas gland cells in the swim bladder wall enables key physiological functions including buoyancy regulation in the face of different hydrostatic pressures. Specific gas gland cells produce and secrete acidic metabolites into the blood in order to reduce the physical solubility of gases and blood gas transport capacity for regulating the volume of the swim bladder. Transcriptomic analyses have provided evidence at the RNA level but no specific studies at the protein level have been carried out so far. Herein, it was the aim of the study to show swim bladder proteins of the yellow stage European eel by label-free LCMS (Q-Exactive Plus) that resulted in the identification of 6223 protein groups. Neurotransmitter receptors and transporters were enriched in the membrane fraction and enzymes for acid production were observed. The list of identified proteins may represent a useful tool for further proteomics experiments on this organ. All MS proteomics data are available at the PRIDE repository with the dataset identifier PXD007850.

Intra-nasal dopamine alleviates cognitive deficits in tgDISC1 rats which overexpress the human DISC1 gene.

The Disrupted-in-Schizophrenia 1 (DISC1) gene has been associated with mental illnesses such as major depression and schizophrenia. The transgenic DISC1 (tgDISC1) rat, which overexpresses the human DISC1 gene, is known to exhibit deficient dopamine (DA) homeostasis. To ascertain whether the DISC1 gene also impacts cognitive functions, 14-15 months old male tgDISC1 rats and wild-type controls were subjected to the novel object preference (NOP) test and the object-based attention test (OBAT) in order to assess short-term memory (1 h), long-term memory (24 h), and attention. RESULTS: The tgDISC1 group exhibited intact short-term memory, but deficient long-term-memory in the NOP test and deficient attention-related behavior in the OBAT. In a different group of tgDISC1 rats, 3 mg/kg intranasally applied dopamine (IN-DA) or its vehicle was applied prior to the NOP or the OBAT test. IN-DA reversed cognitive deficits in both the NOP and OBAT tests. In a further cohort of tgDISC1 rats, post-mortem levels of DA, noradrenaline, serotonin and acetylcholine were determined in a variety of brain regions. The tgDISC1 group had less DA in the neostriatum, hippocampus and amygdala, less acetylcholine in neostriatum, nucleus accumbens, hippocampus, and amygdala, more serotonin in the nucleus accumbens, and less serotonin and noradrenaline in the amygdala. CONCLUSIONS: Our findings show that DISC1 overexpression and misassembly is associated with deficits in long-term memory and attention-related behavior. Since behavioral impairments in tgDISC1 rats were reversed by IN-DA, DA deficiency may be a major cause for the behavioral deficits expressed in this model.

Validation of dopamine receptor DRD1 and DRD2 antibodies using receptor deficient mice.

Dopamine receptors 1 and 2 (DRD1, DRD2) are essential for signaling in the brain for a multitude of brain functions. Previous work using several antibodies against these receptors is abundant but only the minority of antibodies used have been validated and, therefore, the results of these studies remain uncertain. Herein, antibodies against DRD1 (Merck Millipore AB1765P, Santa Cruz Biotechnology sc-14001, Sigma Aldrich D2944, Alomone Labs ADR-001) and DRD2 (Abcam ab21218, Merck Millipore AB5084P, Santa Cruz Biotechnology sc-5303) have been tested using western blotting and immunohistochemistry on mouse striatum (wild type and corresponding knock-out mice) and when specific, they were further evaluated on rat and human striatum. Moreover, a DRD1 antibody and a DRD2 antibody that were found specific in our tests were used for immunoprecipitation with subsequent mass spectrometrical identification of the immunoprecipitate. Two out of nine antibodies (anti DRD1 Sigma Aldrich D2944 and anti DRD2 Merck Millipore AB5084P) against the abovementioned dopamine receptors were specific for DRD1 and DRD2 as evaluated by western blotting and immunohistochemistry and the immunoprecipitate indeed contained DRD1 and DRD2 as revealed by mass spectrometry. The observed findings may question the use of so far non-validated antibodies against the abovementioned dopamine receptors. Own observations may be valuable for the interpretation of previous results and the design of future studies using dopamine receptors DRD1 or DRD2.

Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels.

The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.

Gel-free mass spectrometry analysis of Drosophila melanogaster heads.

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under-represented group in proteomics datasets. Herein, a bottom-up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter-aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed-phase (RP) LC at high pH in the first dimension and acidic RP-LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 (http://proteomecentral.proteomexchange.org/dataset/PXD001712).

Individual phases of contextual fear conditioning differentially modulate dorsal and ventral hippocampal GluA1-3, GluN1-containing receptor complexes and subunits.

In contextual fear conditioning (CFC), the use of pharmacological and lesion approaches has helped to understand that there are differential roles for the dorsal hippocampus (DH) and the ventral hippocampus (VH) in the acquisition, consolidation and retrieval phases. Concomitant analysis of the DH and the VH in individual phases with respect to α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptor subtype N1 (GluN1)-containing complexes (RCC) and subunits has not been reported so far. Herein, CFC was performed in mice that were euthanized at different time points. DH and VH samples were taken for the determination of RCC and subunit levels using BN- and SDS-PAGE, respectively, with subsequent Western blotting. Evaluation of spine densities, morphology, and immunohistochemistry of GluA1 and GluA2 was performed. In the acquisition phase levels of GluA1-RCC and subunits in VH were increased. In the consolidation phase GluA1- and GluA2-RCC levels were increased in DH and VH, while both receptor subunit levels were increased in the VH only. In the retrieval phase GluA1-RCC, subunits thereof and GluA2-RCC were increased in DH and VH, whereas GluA2 subunits were increased in the VH only. GluN1-RCC levels were increased in acquisition and consolidation phase, while subunit levels in the acquisition phase were increased only in the DH. The immunohistochemical studies in the individual phases in subareas of hippocampus supported immunochemical changes of GluA1 and GluA2 RCC's. Dendritic spine densities and the prevalence of thin spines in the acquisition phase of VH and mushroom spines in the retrieval phase of the VH and DH were increased. The findings from the current study suggest different receptor and receptor complex patterns in the individual phases in CFC and in DH and VH. The results propose that different RCCs are formed in the individual phases and that VH and DH may be involved in CFC.

Degradation by Design: New Cyclin K Degraders from Old CDK Inhibitors.

Small molecules that induce protein degradation hold the potential to overcome several limitations of the currently available inhibitors. Monovalent or molecular glue degraders, in particular, enable the benefits of protein degradation without the disadvantages of high molecular weight and the resulting challenge in drug development that are associated with bivalent molecules like Proteolysis Targeting Chimeras. One key challenge in designing monovalent degraders is how to build in the degrader activity─how can we convert an inhibitor into a degrader? If degradation activity requires very specific molecular features, it will be difficult to find new degraders and challenging to optimize those degraders toward drugs. Herein, we demonstrate that an unexpectedly wide range of modifications to the degradation-inducing group of the cyclin K degrader CR8 are tolerated, including both aromatic and nonaromatic groups. We used these findings to convert the pan-CDK inhibitors dinaciclib and AT-7519 to Cyclin K degraders, leading to a novel dinaciclib-based compound with improved degradation activity compared to CR8 and confirm the mechanism of degradation. These results suggest that general design principles can be generated for the development and optimization of monovalent degraders.

The Lack of Dopamine Transporter Is Associated With Conditional Associative Learning Impairments and Striatal Proteomic Changes.

Dopamine (DA) is critically involved in different functions of the central nervous system (CNS) including control of voluntary movement, affect, reward, sleep, and cognition. One of the key components of DA neurotransmission is DA reuptake by the DA transporter (DAT), ensuring rapid clearance of DA from the synaptic cleft. Thus, lack of DAT leads to persistent high extracellular DA levels. While there is strong evidence for a role of striatal dopaminergic activity in learning and memory processes, little is known about the contribution of DAT deficiency to conditional learning impairments and underlying molecular processes. DAT-knockout (DAT-KO) rats were tested in a set of behavioral experiments evaluating conditional associative learning, which requires unaltered striatal function. In parallel, a large-scale proteomic analysis of the striatum was performed to identify molecular factors probably underlying behavioral patterns. DAT-KO rats were incapable to acquire a new operant skill in Pavlovian/instrumental autoshaping, although the conditional stimulus-unconditional stimulus (CS-US) association seems to be unaffected. These findings suggest that DAT directly or indirectly contributes to the reduction of transference of incentive salience from the reward to the CS. We propose that specific impairment of conditional learning might be caused by molecular adaptations to the hyperdopaminergic state, presumably by dopamine receptor 1 (DRD1) hypofunction, as proposed by proteomic analysis. Whether DRD1 downregulation can cause cognitive deficits in the hyperdopaminergic state is the subject of discussion, and further studies are needed to answer this question. This study may be useful for the interpretation of previous and the design of future studies in the dopamine field.

Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Dopamine type 1- and 2-like signaling in the modulation of spatial reference learning and memory.

Spatial reference memory is known to be modulated by the dopaminergic system involving different brain regions. Here, we sought to identify the contribution of D1 (D1R) and D2 (D2R)-like dopamine receptor signaling on learning and memory in a food rewarded hole-board task by intracerebroventricular infusing D1R- and D2R- like receptor agonists (SKF-81297 and Sumanirole) and antagonists (SCH 23390 and Remoxipride) once 30 min prior to daily training sessions. D1R agonism induced persistent enhancement of performance, whereas D1R antagonism impaired reference memory formation. D2R agonist and antagonist exerted no effects. Phase specific comparisons revealed an enhancement of spatial acquisition in the presence of the D1R but not D2R agonism on acquisition, but not during retention. Since task difficulty might skew dopamine-induced improvements in learning and memory, we tested the D1R agonist in the hole-board task with increased difficulty. Drug treated animals performed significantly better during all training phases, with results better resolved than in the easy task. Additionally, proteomic analysis of the prefrontal cortex revealed ninety six proteins to be regulated by D1R agonism, from which 35 were correlated with behavioral performance. Obtained targets were grouped by function, showing synaptic transmission, synaptic remodeling, and dendritic spine morphology as the major functional classes affected. In sum, we find that activation of D1R signaling during spatial acquisition and retention improved reference memory index, depended on the task difficulty, and altered the proteome landscape of the prefrontal cortex indicative of massive organizational synaptic restructuring.

A novel role for NUPR1 in the keratinocyte stress response to UV oxidized phospholipids.

Ultraviolet light is the dominant environmental oxidative skin stressor and a major skin aging factor. We studied which oxidized phospholipid (OxPL) mediators would be generated in primary human keratinocytes (KC) upon exposure to ultraviolet A light (UVA) and investigated the contribution of OxPL to UVA responses. Mass spectrometric analysis immediately or 24 h post UV stress revealed significant changes in abundance of 173 and 84 lipid species, respectively. We identified known and novel lipid species including known bioactive and also potentially reactive carbonyl containing species. We found indication for selective metabolism and degradation of selected reactive lipids. Exposure to both UVA and to in vitro UVA - oxidized phospholipids activated, on transcriptome and proteome level, NRF2/antioxidant response signaling, lipid metabolizing enzyme expression and unfolded protein response (UPR) signaling. We identified NUPR1 as an upstream regulator of UVA/OxPL transcriptional stress responses and found this protein to be expressed in the epidermis. Silencing of NUPR1 resulted in augmented expression of antioxidant and lipid detoxification genes and disturbed the cell cycle, making it a potential key factor in skin reactive oxygen species (ROS) responses intimately involved in aging and pathology.

A proteotranscriptomic study of silk-producing glands from the orb-weaving spiders.

Orb-weaving spiders can produce different silk fibers, which constitute outstanding materials characterized by their high strength and elasticity. Researchers have tried to reproduce the fibers of these proteins synthetically and/or by using recombinant DNA technology, but only a few of the natural physicochemical and biophysical properties have been obtained to date. Female orb-web-spiders present seven silk-glands, which synthesize the spidroins and a series of other proteins, which interact with the spidroins, resulting in silk fibers with notable physicochemical properties. Despite the recognized importance of the silk-glands for understanding how the fibers are produced and processed, the investigation of these glands is at a nascent stage. In the current study we present the assembled transcriptome of silk-producing glands from the orb-weaving spider Nephila clavipes, as well as develop a large-scale proteomic approach for in-depth analyses of silk-producing glands. The present investigation revealed an extensive repertoire of hitherto undescribed proteins involved in silk secretion and processing, such as prevention of degradation during the silk spinning process, transportation, protection against proteolytic autolysis and against oxidative stress, molecular folding and stabilization, and post-translational modifications. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among three groups of silk-producing organisms - (i) Araneomorphae spiders, (ii) Mygalomorphae spiders, and (iii) silk-producing insects. A common orthologous gene, which was annotated as silk gland factor-3 is present among all species analysed. This protein belongs to a transcription factor family, that is important and related to the development of the silk apparatus synthesis in the silk glands of silk-producing arthropods.

Quantitative Proteomics of Synaptosomal Fractions in a Rat Overexpressing Human DISC1 Gene Indicates Profound Synaptic Dysregulation in the Dorsal Striatum.

Disrupted-in-schizophrenia 1 (DISC1) is a key protein involved in behavioral processes and various mental disorders, including schizophrenia and major depression. A transgenic rat overexpressing non-mutant human DISC1, modeling aberrant proteostasis of the DISC1 protein, displays behavioral, biochemical and anatomical deficits consistent with aspects of mental disorders, including changes in the dorsal striatum, an anatomical region critical in the development of behavioral disorders. Herein, dorsal striatum of 10 transgenic DISC1 (tgDISC1) and 10 wild type (WT) littermate control rats was used for synaptosomal preparations and for performing liquid chromatography-tandem mass spectrometry (LC-MS)-based quantitative proteomics, using isobaric labeling (TMT10plex). Functional enrichment analysis was generated from proteins with level changes. The increase in DISC1 expression leads to changes in proteins and synaptic-associated processes including membrane trafficking, ion transport, synaptic organization and neurodevelopment. Canonical pathway analysis assigned proteins with level changes to actin cytoskeleton, Gαq, Rho family GTPase and Rho GDI, axonal guidance, ephrin receptor and dopamine-DARPP32 feedback in cAMP signaling. DISC1-regulated proteins proposed in the current study are also highly associated with neurodevelopmental and mental disorders. Bioinformatics analyses from the current study predicted that the following biological processes may be activated by overexpression of DISC1, i.e., regulation of cell quantities, neuronal and axonal extension and long term potentiation. Our findings demonstrate that the effects of overexpression of non-mutant DISC1 or its misassembly has profound consequences on protein networks essential for behavioral control. These results are also relevant for the interpretation of previous as well as for the design of future studies on DISC1.