Evgenij Fiskin

Fluorescence-Based Sensors to Monitor Localization and Functions of Linear and K63-Linked Ubiquitin Chains in Cells

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in vitro and in vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in vivo.

Posttranscriptional destabilization of the liver‐specific long noncoding RNA HULC by the IGF2 mRNA‐binding protein 1 (IGF2BP1)

Selected long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis. Although the cellular functions of these transcripts can be diverse, many lncRNAs regulate gene expression. In contrast, factors that control the expression of lncRNAs remain largely unknown. Here we investigated the impact of RNA binding proteins on the expression of the liver cancer‐associated lncRNA HULC (highly up‐regulated in liver cancer). First, we validated the strong up‐regulation of HULC in human hepatocellular carcinoma. To elucidate posttranscriptional regulatory mechanisms governing HULC expression, we applied an RNA affinity purification approach to identify specific protein interaction partners and potential regulators. This method identified the family of IGF2BPs (IGF2 mRNA‐binding proteins) as specific binding partners of HULC. Depletion of IGF2BP1, also known as IMP1, but not of IGF2BP2 or IGF2BP3, led to an increased HULC half‐life and higher steady‐state expression levels, indicating a posttranscriptional regulatory mechanism. Importantly, HULC represents the first IGF2BP substrate that is destabilized. To elucidate the mechanism by which IGF2BP1 destabilizes HULC, the CNOT1 protein was identified as a novel interaction partner of IGF2BP1. CNOT1 is the scaffold of the human CCR4‐NOT deadenylase complex, a major component of the cytoplasmic RNA decay machinery. Indeed, depletion of CNOT1 increased HULC half‐life and expression. Thus, IGF2BP1 acts as an adaptor protein that recruits the CCR4‐NOT complex and thereby initiates the degradation of the lncRNA HULC. Conclusion: Our findings provide important insights into the regulation of lncRNA expression and identify a novel function for IGF2BP1 in RNA metabolism. (Hepatology 2013;58:1703–1712)

Structural basis for phosphorylation-triggered autophagic clearance of Salmonella

Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg¹¹ and Lys⁵¹ in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an important protumorigenic factor in hepatocellular carcinoma

Hepatocarcinogenesis is a stepwise process. It involves several genetic and epigenetic alterations, e.g., loss of tumor suppressor gene expression (TP53, PTEN, RB) as well as activation of oncogenes (c‐MYC, MET, BRAF, RAS). However, the role of RNA‐binding proteins (RBPs), which regulate tumor suppressor and oncogene expression at the posttranscriptional level, are not well understood in hepatocellular carcinoma (HCC). Here we analyzed RBPs induced in human liver cancer, revealing 116 RBPs with a significant and more than 2‐fold higher expression in HCC compared to normal liver tissue. We focused our subsequent analyses on the Insulin‐like growth factor 2 messenger RNA (mRNA)‐binding protein 1 (IGF2BP1) representing the most strongly up‐regulated RBP in HCC in our cohort. Depletion of IGF2BP1 from multiple liver cancer cell lines inhibits proliferation and induces apoptosis in vitro. Accordingly, murine xenograft assays after stable depletion of IGF2BP1 reveal that tumor growth, but not tumor initiation, strongly depends on IGF2BP1 in vivo. At the molecular level, IGF2BP1 binds to and stabilizes the c‐MYC and MKI67 mRNAs and increases c‐Myc and Ki‐67 protein expression, two potent regulators of cell proliferation and apoptosis. These substrates likely mediate the impact of IGF2BP1 in human liver cancer, but certainly additional target genes contribute to its function. Conclusion: The RNA‐binding protein IGF2BP1 is an important protumorigenic factor in liver carcinogenesis. Hence, therapeutic targeting of IGF2BP1 may offer options for intervention in human HCC. (Hepatology 2014;59:1900–1911)

Global Analysis of Host and Bacterial Ubiquitinome in Response to Salmonella Typhimurium Infection.

Ubiquitination serves as a critical signal in the host immune response to infection. Many pathogens have evolved strategies to exploit the ubiquitin (Ub) system to promote their own survival through a complex interplay between host defense machinery and bacterial virulence factors. Here we report dynamic changes in the global ubiquitinome of host epithelial cells and invading pathogen in response to Salmonella Typhimurium infection. The most significant alterations in the host ubiquitinome concern components of the actin cytoskeleton, NF-κB and autophagy pathways, and the Ub and RHO GTPase systems. Specifically, infection-induced ubiquitination promotes CDC42 activity and linear ubiquitin chain formation, both being required for NF-κB activation. Conversely, the bacterial ubiquitinome exhibited extensive ubiquitination of various effectors and several outer membrane proteins. Moreover, we reveal that bacterial Ub-modifying enzymes modulate a unique subset of host targets, affecting different stages of Salmonella infection.

Bacteria-host relationship: ubiquitin ligases as weapons of invasion

Eukaryotic cells utilize the ubiquitin (Ub) system for maintaining a balanced functioning of cellular pathways. Although the Ub system is exclusive to eukaryotes, prokaryotic bacteria have developed an armory of Ub ligase enzymes that are capable of employing the Ub systems of various hosts, ranging from plant to animal cells. These enzymes have been acquired through the evolution and can be classified into three main classes, RING (really interesting new gene), HECT (homologous to the E6-AP carboxyl terminus) and NEL (novel E3 ligases). In this review we describe the roles played by different classes of bacterial Ub ligases in infection and pathogenicity. We also provide an overview of the different mechanisms by which bacteria mimic specific components of the host Ub system and outline the gaps in our current understanding of their functions. Additionally, we discuss approaches and experimental tools for validating this class of enzymes as potential novel antibacterial therapy targets.

A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression

Abstract Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage. Our analysis revealed two pairs of mRNA-lncRNA sense-antisense transcripts being inversely expressed upon DNA damage. The lncRNA NOP14-AS1 was strongly upregulated upon DNA damage, while the mRNA for NOP14 was downregulated, both in a p53-dependent manner. For another pair, the lncRNA LIPE-AS1 was downregulated, while its antisense mRNA CEACAM1 was upregulated. To test whether as expected the antisense genes would regulate each other resulting in this highly significant inverse correlation, we employed antisense oligonucleotides and RNAi to study transcript-dependent effects as well as dCas9-based transcriptional modulation by CRISPRi/CRISPRa for transcription-dependent effects. Surprisingly, despite the strong stimulus-dependent inverse correlation, our data indicate that neither transcript- nor transcription-dependent mechanisms explain the inverse regulation of NOP14-AS1:NOP14 or LIPE-AS1:CEACAM1 expression. Hence, sense-antisense pairs whose expression is strongly—positively or negatively—correlated can be nonetheless regulated independently. This highlights the requirement of individual experimental studies for each antisense pair and prohibits drawing conclusions on regulatory mechanisms from expression correlations.

Structural basis for the recognition and degradation of host TRIM proteins by Salmonella effector SopA.

The hallmark of Salmonella Typhimurium infection is an acute intestinal inflammatory response, which is mediated through the action of secreted bacterial effector proteins. The pro-inflammatory Salmonella effector SopA is a HECT-like E3 ligase, which was previously proposed to activate host RING ligases TRIM56 and TRIM65. Here we elucidate an inhibitory mechanism of TRIM56 and TRIM65 targeting by SopA. We present the crystal structure of SopA in complex with the RING domain of human TRIM56, revealing the atomic details of their interaction and the basis for SopA selectivity towards TRIM56 and TRIM65. Structure-guided biochemical analysis shows that SopA inhibits TRIM56 E3 ligase activity by occluding the E2-interacting surface of TRIM56. We further demonstrate that SopA ubiquitinates TRIM56 and TRIM65, resulting in their proteasomal degradation during infection. Our results provide the basis for how a bacterial HECT ligase blocks host RING ligases and exemplifies the multivalent power of bacterial effectors during infection.

Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain–specific sensors

Despite intensive research, there is a distinct lack of methodology for visualizing endogenous ubiquitination in living cells. In this protocol, we describe how unique properties of ubiquitin (Ub)-binding domains (UBDs) can be used to selectively detect, visualize and inhibit Ub-dependent processes in mammalian cells. The procedure deals with designing and validating the binding selectivity of GFP-tagged K63- and linear-linked sensors (TAB2 NZF and NEMO UBAN, respectively) in vitro. We describe how these moieties can be used to inhibit tumor necrosis factor (TNF)-mediated NF-κB signaling and to detect ubiquitinated cytosolic Salmonella in living cells, emphasizing a more flexible use compared with chain-specific antibodies. These chain-specific sensors can be used to detect Ub-like or autophagy-related modifiers and, in combination with mass spectrometry, to identify new Ub targets. These Ub (-like) sensors can be designed, constructed and tested in ∼2–3 weeks.

Parkin promotes cell survival via linear ubiquitination.

Mitochondrial dysfunction has been linked to the pathogenesis of Parkinsons disease (PD). PD‐associated mutants of Parkin show defects in mitophagy, suggesting that Parkin exerts its neuroprotective role by removal of damaged mitochondria. Muller‐Rischart et al (2013) now uncover an autophagy‐independent pathway involving Parkin‐mediated activation of the LUBAC ubiquitin ligase to prevent mitochondrial impairment and thereby to promote survival. PD represents the most common movement disorder and is caused by dopaminergic neurodegeneration. As the majority of cases arise sporadically, the identification of monogenetic variants marked the beginning of a new era in PD research. Loss‐of‐function mutations mapped to the Parkin ubiquitin ligase and PINK1 kinase loci were shown to cause autosomal recessive juvenile parkinsonism (Kitada et al , 1998). The generation of PINK1 and Parkin deletion mutants in Drosophila illustrated their requirement for the maintenance of mitochondrial integrity (Clark et al , 2006) and supported the notion that mitochondrial dysfunction is a major etiological factor in PD. These reports also demonstrated the genetic epistasis of PINK1 and Parkin, placing them in a common or converging pathway. Further studies revealed their involvement in the autophagic removal of damaged mitochondria (Narendra et al , 2008). PINK1 is selectively retained and stabilized at the outer membrane of depolarized mitochondria and serves as a signal for Parkin recruitment and activation. Subsequent ubiquitination of mitochondrial proteins by Parkin drives affected mitochondria into autophagosomes (Geisler et al , 2010). In addition, mounting evidence …