Highly specific intracellular ubiquitination of a small molecule.

Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here, we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains causing broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent upon two homologous E3 ubiquitin ligases, RNF19A and RNF19B. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule, and more broadly raises the potential for posttranslational modification in trans.

Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance.

Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8+ and CD4+ T cells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment.

Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine.

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.

Fingolimod effects on the brain are mediated through biochemical modulation of bioenergetics, autophagy, and neuroinflammatory networks.

Fingolimod (FTY720) is an oral drug approved by the Food and Drug Administration (FDA) for management of multiple sclerosis (MS) symptoms, which has also shown beneficial effects against Alzheimer's (AD) and Parkinson's (PD) diseases pathologies. Although an extensive effort has been made to identify mechanisms underpinning its therapeutic effects, much remains unknown. Here, we investigated Fingolimod induced proteome changes in the cerebellum (CB) and frontal cortex (FC) regions of the brain which are known to be severely affected in MS, using a tandem mass tag (TMT) isobaric labeling-based quantitative mass-spectrometric approach to investigate the mechanism of action of Fingolimod. This study identified 6749 and 6319 proteins in CB and FC, respectively, and returned 2609 and 3086 differentially expressed proteins in mouse CB and FC, respectively, between Fingolimod treated and control groups. Subsequent bioinformatics analyses indicated a metabolic reprogramming in both brain regions of the Fingolimod treated group, where oxidative phosphorylation was upregulated while glycolysis and pentose phosphate pathway were downregulated. In addition, modulation of neuroinflammation in the Fingolimod treated group was indicated by upregulation of retrograde endocannabinoid signaling and autophagy pathways, and downregulation of neuroinflammation related pathways including neutrophil degranulation and the IL-12 mediated signaling pathway. Our findings suggest that Fingolimod may exert its protective effects on the brain by inducing metabolic reprogramming and neuroinflammation pathway modulation.

Genome-wide transcript and protein analysis highlights the role of protein homeostasis in the aging mouse heart.

Investigation of the molecular mechanisms of aging in the human heart is challenging because of confounding factors, such as diet and medications, as well as limited access to tissues from healthy aging individuals. The laboratory mouse provides an ideal model to study aging in healthy individuals in a controlled environment. However, previous mouse studies have examined only a narrow range of the genetic variation that shapes individual differences during aging. Here, we analyze transcriptome and proteome data from 185 genetically diverse male and female mice at ages 6, 12, and 18 mo to characterize molecular changes that occur in the aging heart. Transcripts and proteins reveal activation of pathways related to exocytosis and cellular transport with age, whereas processes involved in protein folding decrease with age. Additional changes are apparent only in the protein data including reduced fatty acid oxidation and increased autophagy. For proteins that form complexes, we see a decline in correlation between their component subunits with age, suggesting age-related loss of stoichiometry. The most affected complexes are themselves involved in protein homeostasis, which potentially contributes to a cycle of progressive breakdown in protein quality control with age. Our findings highlight the important role of post-transcriptional regulation in aging. In addition, we identify genetic loci that modulate age-related changes in protein homeostasis, suggesting that genetic variation can alter the molecular aging process.

Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney.

Little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand these changes, we measured mRNA and protein levels in genetically diverse mice at different ages. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated.

Retinal proteomics of experimental glaucoma model reveal intraocular pressure-induced mediators of neurodegenerative changes.

Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3 ± 0.1 vs 20.8 ± 1.6 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using a multiplexed quantitative proteomics approach (TMT-MS3). Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated (≤0.83) while the expression of 109 proteins was upregulated (≥1.2-fold change) under glaucoma conditions (P ≤ .05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton, and actin filament organisation, along with increased expression of proteins in coagulation cascade, apoptosis, oxidative stress, and RNA processing. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport, and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism, and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins were exclusive to human glaucoma. These findings provide insights into the neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP.

Quantitative analysis of Y-Chromosome gene expression across 36 human tissues.

Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes.

Cdk1 Controls Global Epigenetic Landscape in Embryonic Stem Cells.

The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

Kinase-independent function of E-type cyclins in liver cancer.

E-type cyclins (cyclins E1 and E2) are components of the core cell cycle machinery and are overexpressed in many human tumor types. E cyclins are thought to drive tumor cell proliferation by activating the cyclin-dependent kinase 2 (CDK2). The cyclin E1 gene represents the site of recurrent integration of the hepatitis B virus in the pathogenesis of hepatocellular carcinoma, and this event is associated with strong up-regulation of cyclin E1 expression. Regardless of the underlying mechanism of tumorigenesis, the majority of liver cancers overexpress E-type cyclins. Here we used conditional cyclin E knockout mice and a liver cancer model to test the requirement for the function of E cyclins in liver tumorigenesis. We show that a ubiquitous, global shutdown of E cyclins did not visibly affect postnatal development or physiology of adult mice. However, an acute ablation of E cyclins halted liver cancer progression. We demonstrated that also human liver cancer cells critically depend on E cyclins for proliferation. In contrast, we found that the function of the cyclin E catalytic partner, CDK2, is dispensable in liver cancer cells. We observed that E cyclins drive proliferation of tumor cells in a CDK2- and kinase-independent mechanism. Our study suggests that compounds which degrade or inhibit cyclin E might represent a highly selective therapeutic strategy for patients with liver cancer, as these compounds would selectively cripple proliferation of tumor cells, while sparing normal tissues.

Age-related neurodegenerative disease associated pathways identified in retinal and vitreous proteome from human glaucoma eyes.

ABSTARCT: Glaucoma is a chronic disease that shares many similarities with other neurodegenerative disorders of the central nervous system. This study was designed to evaluate the association between glaucoma and other neurodegenerative disorders by investigating glaucoma-associated protein changes in the retina and vitreous humour. The multiplexed Tandem Mass Tag based proteomics (TMT-MS3) was carried out on retinal tissue and vitreous humour fluid collected from glaucoma patients and age-matched controls followed by functional pathway and protein network interaction analysis. About 5000 proteins were quantified from retinal tissue and vitreous fluid of glaucoma and control eyes. Of the differentially regulated proteins, 122 were found linked with pathophysiology of Alzheimer's disease (AD). Pathway analyses of differentially regulated proteins indicate defects in mitochondrial oxidative phosphorylation machinery. The classical complement pathway associated proteins were activated in the glaucoma samples suggesting an innate inflammatory response. The majority of common differentially regulated proteins in both tissues were members of functional protein networks associated brain changes in AD and other chronic degenerative conditions. Identification of previously reported and novel pathways in glaucoma that overlap with other CNS neurodegenerative disorders promises to provide renewed understanding of the aetiology and pathogenesis of age related neurodegenerative diseases.

The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival.

D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.

G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells.

Progression of mammalian cells through the G1 and S phases of the cell cycle is driven by the D-type and E-type cyclins. According to the current models, at least one of these cyclin families must be present to allow cell proliferation. Here, we show that several cell types can proliferate in the absence of all G1 cyclins. However, following ablation of G1 cyclins, embryonic stem (ES) cells attenuated their pluripotent characteristics, with the majority of cells acquiring the trophectodermal cell fate. We established that G1 cyclins, together with their associated cyclin-dependent kinases (CDKs), phosphorylate and stabilize the core pluripotency factors Nanog, Sox2 and Oct4. Treatment of murine ES cells, patient-derived glioblastoma tumour-initiating cells, or triple-negative breast cancer cells with a CDK inhibitor strongly decreased Sox2 and Oct4 levels. Our findings suggest that CDK inhibition might represent an attractive therapeutic strategy by targeting glioblastoma tumour-initiating cells, which depend on Sox2 to maintain their tumorigenic potential.

Defining the consequences of genetic variation on a proteome-wide scale.

Genetic variation modulates protein expression through both transcriptional and post-transcriptional mechanisms. To characterize the consequences of natural genetic diversity on the proteome, here we combine a multiplexed, mass spectrometry-based method for protein quantification with an emerging outbred mouse model containing extensive genetic variation from eight inbred founder strains. By measuring genome-wide transcript and protein expression in livers from 192 Diversity outbred mice, we identify 2,866 protein quantitative trait loci (pQTL) with twice as many local as distant genetic variants. These data support distinct transcriptional and post-transcriptional models underlying the observed pQTL effects. Using a sensitive approach to mediation analysis, we often identified a second protein or transcript as the causal mediator of distant pQTL. Our analysis reveals an extensive network of direct protein-protein interactions. Finally, we show that local genotype can provide accurate predictions of protein abundance in an independent cohort of collaborative cross mice.

Blood biomarkers in a mouse model of CADASIL.

Mutations in NOTCH 3 are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a neurological disorder characterized by stroke, and vascular cognitive impairment and dementia. Loss of vascular smooth muscle cells (VSMC) and accumulation of granular osmiophilic material (GOM) deposits are hallmarks of CADASIL. There are no therapies for CADASIL and experimental endpoints to examine the preclinical efficacy of potential drugs are lacking. This study aims to use a mouse carrying the C455R mutation in Notch 3 to identify biomarkers associated with CADASIL. Mass spectrometry and antibody arrays were used to explore the aorta and blood proteomes of CADASIL mice, ELISA assays were utilized for biomarker validation, a ligand-dependent assay was applied to examine the relationship between Notch signaling and biomarker expression, and retinal histology was performed for quantification of VSMC loss in arteries. Two-hundred day-old mice with the C455R CADASIL mutation in Notch 3 mice display robust VSMC loss in retinal arteries and had increased plasma levels of collagen18α1/endostatin (col18α1) and high-temperature requirement A serine peptidase 1 (HTRA1) and reduced levels of Notch 3 extracellular domain (N3ECD), compared to control wild type mice. Measurements of plasma endostatin, HTRA1 and N3ECD, along with VSMC quantification in retinal arteries, may serve as surrogate endpoints for assessing efficacy in preclinical therapeutic studies of CADASIL using mice.

Quantitative proteomic analysis of two different rice varieties reveals that drought tolerance is correlated with reduced abundance of photosynthetic machinery and increased abundance of ClpD1 protease.

UNLABELLED: Rice is the major staple food for more than half of world's population. As global climate changes, we are observing more floods, droughts and severe heat waves. Two rice cultivars with contrasting genetic backgrounds and levels of tolerance to drought, Nipponbare and IAC1131, were used in this study. Four-week-old seedlings of both cultivars were grown in large soil volumes and then exposed to moderate and extreme drought for 7days, followed by 3days of re-watering. Mature leaves were harvested from plants from each treatment for protein extraction and subsequent shotgun proteomic analysis, with validation of selected proteins by western blotting. Gene Ontology (GO) annotations of differentially expressed proteins provide insights into the metabolic pathways that are involved in drought stress resistance. Our data indicate that IAC1131 appears to be better able to cope with stressful conditions by upregulating a suite of stress and defence response related proteins. Nipponbare, in contrast, lacks the range of stress responses shown by the more stress tolerant variety, and responds to drought stress by initiating a partial shutdown of chlorophyll biosynthesis in an apparent attempt to preserve resources. SIGNIFICANCE: In this study, two rice genotypes with contrasting drought tolerance were exposed to soil water deficits, and proteomic changes were observed in mature leaf laminae. Plants were well watered and then switched to conditions of either moderate drought or extreme drought followed by three days of recovery. Proteins were identified and quantified using both label-free and Tandem Mass Tag multiplexing approaches. Several biochemical pathways were significantly altered in response to water deficit. Most notably, the up-regulation of ClpD1 protease responded strongly in the drought-tolerant landrace; this protein is typically involved in heat and osmotic stress response. In contrast, porphyrin and chlorophyll biosynthesis pathways were down-regulated, indicating suppression of the photosynthetic machinery.

Induction of virulence factors in Giardia duodenalis independent of host attachment.

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response.