Discovery of pyrazolo[3,4-d]pyrimidines as novel mitogen-activated protein kinase kinase 3 (MKK3) inhibitors.

The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.

Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits.

The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1's substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1's intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD's role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

Post-Translational Modifications and RNA-Binding Proteins.

RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.

The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation.

Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.

Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy.

Autophagy, the process by which proteins or organelles are engulfed by autophagosomes and delivered for vacuolar/lysosomal degradation, is induced to ensure survival under starvation and other stresses. A selective autophagic pathway for 60S ribosomal subunits elicited by nitrogen starvation in yeast-ribophagy-was recently described and requires the Ubp3-Bre5 deubiquitylating enzyme. This discovery implied that an E3 ligases act upstream, whether inhibiting the process or providing an initial required signal. In this paper, we show that Ltn1/Rkr1, a 60S ribosome-associated E3 implicated in translational surveillance, acts as an inhibitor of 60S ribosomal subunit ribophagy and is antagonized by Ubp3. The ribosomal protein Rpl25 is a relevant target. Its ubiquitylation is Ltn1 dependent and Ubp3 reversed, and mutation of its ubiquitylation site rendered ribophagy less dependent on Ubp3. Consistently, the expression of Ltn1-but not Ubp3-rapidly decreased after starvation, presumably to allow ribophagy to proceed. Thus, Ltn1 and Ubp3-Bre5 likely contribute to adapt ribophagy activity to both nutrient supply and protein translation.

Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase.

Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.

Downregulation of VAPB expression in motor neurons derived from induced pluripotent stem cells of ALS8 patients.

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope, since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but, in contrast to over-expression systems, cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8, in agreement with the observed reduction of VAPB in sporadic ALS.

Role of a ribosome-associated E3 ubiquitin ligase in protein quality control.

Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How eukaryotic cells eliminate non-stop proteins has remained unknown. Here we show that the Saccharomyces cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins, in a process that is mechanistically distinct but conceptually analogous to that performed by ssrA: Ltn1 is predominantly associated with ribosomes, and it marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue Listerin.

Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor.

The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.

Identification of novel differentially expressed genes in human astrocytomas by cDNA representational difference analysis.

Diffuse infiltrating gliomas are the most common tumors of the central nervous system (CNS), naturally progressing from a lower-grade to a higher-grade malignancy. Several genetic alterations have been correlated with astrocytic tumors; however, a number of as yet unknown genes may also be involved. Therefore, we set out to search for genes that are differentially expressed in anaplastic astrocytoma and normal CNS tissue by applying a PCR-based subtractive hybridization approach, namely, representational difference analysis (RDA). The results of DNA sequencing of a sample (96 cDNA clones) from the subtracted library allowed the identification of 18 different genes, some of which were represented by several cDNA clones, coding for the Np95, LMO1, FCGBP, DSCAM, and taxilin proteins. Quantitative real-time PCR analysis for five of these genes was performed using samples of astrocytic tumors of different grades, confirming their higher expression when compared to non-tumoral CNS tissue. Identification of differentially expressed genes present in gliomas but not in normal CNS tissue is important not only to better understand the molecular basis of these cancers, but also to generate diagnostic DNA chips, which may be useful in future therapeutic intervention.

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer.

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.