Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we ...show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.
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•Keap1 loss and Nrf2 activation induce metastasis in LUAD by accumulating Bach1•Nrf2 inhibits the Fbxo22-mediated degradation of Bach1 in a Ho1-dependent manner•Bach1 signature is associated with metastasis and shortened survival in LUAD patients•Ho1 inhibitors reduce LUAD metastasis in a Fbxo22- and Bach1-dependent manner
Stabilization of the transcription factor Bach1 drives metastasis of lung adenocarcinoma and this can be counteracted by the pharmacological inhibition of heme oxygenase.
Proteomic studies of human Alzheimer's disease brain tissue have potential to identify protein changes that drive disease, and to identify new drug targets. Here, we analyse 38 published Alzheimer's ...disease proteomic studies, generating a map of protein changes in human brain tissue across thirteen brain regions, three disease stages (preclinical Alzheimer's disease, mild cognitive impairment, advanced Alzheimer's disease), and proteins enriched in amyloid plaques, neurofibrillary tangles, and cerebral amyloid angiopathy. Our dataset is compiled into a searchable database (NeuroPro). We found 848 proteins were consistently altered in 5 or more studies. Comparison of protein changes in early-stage and advanced Alzheimer's disease revealed proteins associated with synapse, vesicle, and lysosomal pathways show change early in disease, but widespread changes in mitochondrial associated protein expression change are only seen in advanced Alzheimer's disease. Protein changes were similar for brain regions considered vulnerable and regions considered resistant. This resource provides insight into Alzheimer's disease brain protein changes and highlights proteins of interest for further study.
Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a ...significant proportion of patients do not respond to anti–PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.
There is a trend in the analysis of shotgun proteomics data that aims to combine information from multiple search engines to increase the number of peptide annotations in an experiment. Typically, ...the degree of search engine complementarity and search engine agreement is visually illustrated by means of Venn diagrams that present the findings of a database search on the level of the nonredundant peptide annotations. We argue this practice to be not fit‐for‐purpose since the diagrams do not take into account and often conceal the information on complementarity and agreement at the level of the spectrum identification. We promote a new type of visualization that provides insight on the peptide sequence agreement at the level of the peptide‐spectrum match (PSM) as a measure of consensus between two search engines with nominal outcomes. We applied the visualizations and percentage sequence agreement to an in‐house data set of our benchmark organism, Caenorhabditis elegans, and illustrated that when assessing the agreement between search engine, one should disentangle the notion of PSM confidence and PSM identity. The visualizations presented in this manuscript provide a more informative assessment of pairs of search engines and are made available as an R function in the Supporting Information.
Tuberous sclerosis complex (TSC) and some focal cortical dysplasias (FCDs) are associated with dysfunctional mTOR signaling, resulting in increased cell growth and ribosomal S6 protein ...phosphorylation (phospho-S6). mTOR inhibitors can reduce TSC tumor growth and seizure frequency, and preclinical FCD studies indicate seizure suppression. This pilot study evaluated safety of mTOR inhibitor everolimus in treatment resistant (failure of >2 anti-seizure medications) TSC and FCD patients undergoing surgical resection and to assess mTOR signaling and molecular pathways.
We evaluated everolimus in 14 treatment resistant epilepsy patients undergoing surgical resection (4.5 mg/m2 daily for 7 days; n = 4 Active, mean age 18.3 years, range 4-26; n = 10, Control, mean age 13.1, range 3-45). Everolimus was well tolerated. Mean plasma everolimus in Active participants were in target range (12.4 ng/ml). Brain phospho-S6 was similar in Active and Control participants with a lower trend in Active participants, with Ser235/236 1.19-fold (p = 0.67) and Ser240/244 1.15-fold lower (p = 0.66). Histologically, Ser235/236 was 1.56-fold (p = 0.37) and Ser240/244 was 5.55-fold lower (p = 0.22). Brain proteomics identified 11 proteins at <15% false discovery rate associated with coagulation system (p = 1.45x10-9) and acute phase response (p = 1.23x10-6) activation. A weighted gene correlation network analysis (WGCNA) of brain proteomics and phospho-S6 identified 5 significant modules. Higher phospho-S6 correlated negatively with cellular respiration and synaptic transmission and positively with organophosphate metabolic process, nuclear mRNA catabolic process, and neuron ensheathment. Brain metabolomics identified 14 increased features in Active participants, including N-acetylaspartylglutamic acid. Plasma proteomics and cytokine analyses revealed no differences.
Short-term everolimus before epilepsy surgery in TSC and FCD resulted in no adverse events and trending lower mTOR signaling (phospho-S6). Future studies should evaluate implications of our findings, including coagulation system activation and everolimus efficacy in FCD, in larger studies with long-term treatment to better understand molecular and clinical effects.
ClinicalTrials.gov NCT02451696.
Data sharing in the field of MS has advanced greatly thanks to innovations such as the standardized formats, data repositories, and publications guidelines. However, there is currently no data ...sharing mechanism that enables real‐time data browsing and deep linking on a large scale: unrestricted data access (particularly at the quantitative level) ultimately requires the user to download a local copy of the relevant data files (e.g., in order to generate extracted ion chromatograms XICs). In this technical resource, we present a set of technologies (collectively termed OpenSlice) that enable the user to quantitatively query hundreds of hours of proteomics discovery data (i.e., nontargeted acquisition) in real time: the user is able to effectively generate XICs for arbitrary masses on the fly and across the entire dataset (so‐called global ion chromatograms), interacting with the results through a very intuitive browser‐based interface. A key design consideration underlying the OpenSlice approach is the notion that every aspect of the acquired data must be accessible through a RESTful uniform resource locator based application programming interface, up to and including individual chromatographic peaks (hence HyperPeaks). A publicly accessible demonstration of this technology based on the Clinical Proteomics Tumor Analysis Consortium CompRef dataset is made available at http://compref.fenyolab.org.
Toxins are detected in sporadic species along the evolutionary tree of the animal kingdom. Venomous animals include scorpions, snakes, bees, wasps, frogs and numerous animals living in the sea such ...as the stonefish, snail, jellyfish, hydra and more. Interestingly, proteins that share a common scaffold with animal toxins also exist in non-venomous species. However, due to their short length and primary sequence diversity, these, toxin-like proteins remain undetected by classical search engines and genome annotation tools. We construct a toxin classification machine and web server called ClanTox (Classifier of Animal Toxins) that is based on the extraction of sequence-driven features from the primary protein sequence followed by the application of a classification system trained on known animal toxins. For a given input list of sequences, from venomous or non-venomous settings, the ClanTox system predicts whether each sequence is toxin-like. ClanTox provides a ranked list of positively predicted candidates according to statistical confidence. For each protein, additional information is presented including the presence of a signal peptide, the number of cysteine residues and the associated functional annotations. ClanTox is a discovery-prediction tool for a relatively overlooked niche of toxin-like cell modulators, many of which are therapeutic agent candidates. The ClanTox web server is freely accessible at http://www.clantox.cs.huji.ac.il.
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor with roles in inflammation and tumorigenicity. A fraction of STAT3 localizes in mitochondria, where it augments ...tumorigenesis via regulation of mitochondrial functions, including modulation of respiration and redox status. We show a novel mechanism for mitochondrial STAT3 regulation of redox homeostasis in triple‐negative breast cancer cells. Loss of STAT3 diminished complex I dehydrogenase activity and impaired NAD+ regeneration, leading to impaired expression of glutathione biosynthetic genes and other antioxidant genes. Expressing mitochondrially restricted STAT3 or replenishment of the cellular NAD pool restored antioxidant gene expression, as did complementation of the NADH dehydrogenase activity by expression of the STAT3‐independent yeast dehydrogenase, NDI1. These NAD‐regulated processes contributed to malignant phenotypes by promoting clonal cell growth and migration. Proximity interaction and protein pull‐down assays identified three components of complex I that associated with mitochondrial STAT3, providing a potential mechanistic basis for how mitochondrial STAT3 affects complex I activity. Our data document a novel mechanism through which mitochondrial STAT3 indirectly controls antioxidant gene regulation through a retrograde NAD+ signal that is modulated by complex I dehydrogenase activity.
STAT3 interacts with respiratory complex I in mitochondria, leading to enhanced NADH dehydrogenase activity, facilitating efficient regeneration of NAD+ during respiration. NAD+ acts as a retrograde signal linking mitochondrial metabolism to changes in nuclear gene expression, leading to induction of antioxidant genes that contribute to the maintenance of redox balance and malignant cell growth, survival, and migration.
Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary ...testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (
n
= 19) and pediatric control cases (
n
= 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (
p
= 6.3 × 10
–15
,
z
= 4.08) and inhibited EIF2 signaling (
p
= 2.0 × 10
–21
,
z
= − 2.56) in frontal cortex, and activated acute phase response in DG (
p
= 8.5 × 10
–6
,
z
= 2.65) and CA1-3 (
p
= 4.7 × 10
–6
,
z
= 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (
n
= 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (
p
= 2.8 × 10
–5
) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG,
p
= 1.8 × 10
–3
) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex,
p
= 8.8 × 10
–5
) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion,
p
= 1.9 × 10
–3
). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.
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