Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.
TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53β promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53β increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53β is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53β depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53β induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.
TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations.
Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action, and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. Here, we sought for a possible connection between transcription and translation. Employing an unbiased screen of multiple human promoters, we identified a positive effect of TATA box on translation and a general coupling between mRNA expression and translational efficiency. Using a CRISPR-Cas9-mediated approach, genome-wide analyses, and in vitro experiments, we show that the rate of transcription regulates the efficiency of translation. Furthermore, we demonstrate that m6A modification of mRNAs is co-transcriptional and depends upon the dynamics of the transcribing RNAPII. Suboptimal transcription rates lead to elevated m6A content, which may result in reduced translation. This study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mRNAs.
The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.
Glutamate-ammonia ligase (GLUL) belongs to the glutamine synthetase family. It catalyzes the synthesis of glutamine from glutamate and ammonia in an ATP-dependent reaction. Here, we found higher expression of GLUL in the breast cancer patients was associated with larger tumor size and higher level of HER2 expression. In addition, GLUL was heterogeneously expressed in various breast cancer cells. The mRNA and protein expression levels of GLUL in SK-BR-3 cells were obviously higher than that in the other types of breast cancer cells. Results showed GLUL knockdown in SK-BR-3 cells could significantly decrease the proliferation ability. Furthermore, GLUL knockdown markedly inhibited the p38 MAPK and ERK1/ERK2 signaling pathways in SK-BR-3 cells. Thus, GLUL may represent a novel target for selectively inhibiting p38 MAPK and ERK1/ERK2 signaling pathways and the proliferation potential of breast cancer cells. J. Cell. Biochem. 118: 2018-2025, 2017. © 2016 Wiley Periodicals, Inc.
Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits site-specific conjugation of drugs. Compared with other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions. Using a tailored conjugation chemistry, we generated highly stable selenomab-drug conjugates and demonstrated their potency and selectivity in vitro and in vivo. These site-specific antibody-drug conjugates built on a selenocysteine interface revealed broad therapeutic utility in liquid and solid malignancy models.
The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.
Signaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions. The data, spanning multiple pathways and comprising ∼70,000 phosphoprotein and ∼260,000 protein measurements, provide a wealth of testable, context-specific hypotheses, several of which we experimentally validate. Furthermore, the data provide a unique resource for computational methods development, permitting empirical assessment of causal network learning in a complex, mammalian setting.