Truncated Protein Variants Research Articles

Expanding the product portfolio of carbon dioxide and hydrogen-based gas fermentation with an evolved strain of Clostridium carboxidivorans

CO2:H2-based gas fermentation with acetogenic Clostridium species are at an early stage of development. This work exploited the Adaptive Laboratory Evolution technique to improve the growth of C. carboxidivorans P7 on CO2 and H2. An adapted strain with decreased growth lag phase and improved biomass production was obtained. Genomic analysis revealed a conserved frameshift mutation in the catalytic subunit of the hexameric hydrogenase gene. The resulted truncated protein variant, most likely lacking its functionality, suggests that other hydrogenases might be more efficient for H2-based growth of this strain. Furthermore, the adapted strain generated hexanol as primary fermentation product. For the first time, hexanol was produced directly from CO2:H2 blend, achieving the highest maximum productivity reported so far via gas fermentation. Traces of valerate, pentanol, eptanol and octanol were observed in the fermentation broth. The adapted strain shows promising to enrich the product spectrum targetable by future gas fermentation processes.

PLAUR splicing pattern in hereditary angioedema patients’ monocytes and macrophages

The PLAUR gene encodes the urokinase-like plasminogen activator receptor (uPAR) and may undergo alternative splicing. Excluding cassette exons 3, 5 and 6 from the transcript results in truncated protein variants whose precise functions have not been elucidated yet. The PLAUR gene is one of several expressed in myeloid cells, where uPAR participates in different cellular processes, including the contact activation system and kallikrein-kinin system, which play an important role in hereditary angioedema (HAE) pathogenesis. A hypothesis about the PLAUR splicing pattern impact on HAE severity was tested. The RT-PCR quantified by capillary electrophoresis was used. Although no significant difference in alternative transcript frequency was observed between healthy volunteers and HAE patients, a significant increase in all cassette exon inclusion variants was revealed during monocyte-to-macrophage differentiation. PLAUR alternative splicing in monocytes and macrophages neither was different between HAE patients and healthy controls, nor reflected disease severity. However, the results showed an PLAUR splicing pattern was changing during monocyte-to-macrophage differentiation, but the significance of these changes is unknown and awaits future clarification.

Structure and function of SARS-CoV-2 nucleocapsid protein measured using optical tweezers, confocal fluorescence, and AFM.

The nucleocapsid (N) protein, one of four structural proteins expressed by SARS-CoV-2, primarily functions by binding and compacting the ∼30 kilobase viral RNA genome (>10 μm linear length) to enable packaging into viral particles (<100 nm diameter). We use optical tweezers to isolate a single ssDNA substrate and observe the binding and compacting function of N protein in real time. The compaction is a multistep process, with kinetics consistent with initial diffusion-limited binding of free protein to the substrate followed by a sudden stochastically triggered reorganization of the protein-DNA complex into a compacted form. Experiments utilizing truncated protein variants lacking either the ordered N or C terminal domains (NTD and CTD) and the intrinsically disordered linker demonstrate that initial binding is driven by the NTD while the formation of the compacted DNA structure requires the CTD. We further localize the binding of fluorescently labeled protein along the substrate using confocal imaging, showing N protein able to both uniformly coat the entire length of the substrate non specifically and subsequently form dense protein clusters through interprotein oligomerization. Finally, various DNA and RNA substrates of differing lengths and sequences are incubated with either full length or truncated N protein and imaged using AFM. Measurement of these nucleic acid-protein complexes reveal both the binding activity of N protein and its ability to remodel complex, folded nucleic acid structures. Our results help elucidate the biophysical mechanism through which N protein enables efficient packaging of the long vRNA under physiological conditions.

Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.

Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.

Structural domains of SARS-CoV-2 nucleocapsid protein coordinate to compact long nucleic acid substrates.

The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion.

Evolution and Diversity of the TopoVI and TopoVI-like Subunits With Extensive Divergence of the TOPOVIBL subunit.

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

Transcription factor ZmWRKY20 interacts with ZmWRKY115 to repress expression of ZmbZIP111 for salt tolerance in maize.

Maize (Zea mays) is an important cereal crop worldwide. However, its yield and quality are adversely affected by salt stress resulting from soil hypersalinity. Exploring the regulatory mechanisms of stress responses is of vital importance to increase maize seed production. In the present study, we screened ethyl methanesulfonate-induced maize mutants and identified a salt-tolerant mutant. A single base was mutated in ZmWRKY20, leading to the formation of a truncated protein variant. A detailed phenotypic analysis revealed that this mutant had significantly higher resistance to wilting and lower reactive oxygen species levels than the inbred line B73. ZmWRKY20 showed transcriptional activity in yeast and specifically bound W-boxes according to the results of our yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Overexpression of ZmWRKY20 decreased salt tolerance in maize. Transcriptome profiling revealed that ZmWRKY20 overexpression extensively reprogrammed genes involved in regulating defense and oxidation-reduction responses. The results substantiate that ZmWRKY20 is directly targeted to the basic leucine zipper (bZIP) motif in the transcription factor ZmbZIP111. It was also verified that ZmWRKY20 interacts with ZmWRKY115 and both proteins act jointly to enhance ZmbZIP111 repression. The results indicate that the ZmWRKY20 and ZmWRKY115 transcription factors interact in the nucleus, leading to repression of ZmbZIP111 expression by directly binding its promoter, and increase the sensitivity of maize seedlings to salt stress. The current study improves our understanding of the complicated responses of maize to salt stress.

A Medicago truncatula SWEET transporter implicated in arbuscule maintenance during arbuscular mycorrhizal symbiosis.

Plants form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which facilitates the acquisition of scarce minerals from the soil. In return, the host plants provide sugars and lipids to its fungal partner. However, the mechanism by which the AM fungi obtain sugars from the plant has remained elusive. In this study we investigated the role of potential SWEET family sugar exporters in AM symbiosis in Medicago truncatula. We show that M.truncatula SWEET1b transporter is strongly upregulated in arbuscule-containing cells compared to roots and localizes to the peri-arbuscular membrane, across which nutrient exchange takes place. Heterologous expression of MtSWEET1b in a yeast hexose transport mutant showed that it mainly transports glucose. Overexpression of MtSWEET1b in M.truncatula roots promoted the growth of intraradical mycelium during AM symbiosis. Surprisingly, two independent Mtsweet1b mutants, which are predicted to produce truncated protein variants impaired in glucose transport, exhibited no significant defects in AM symbiosis. However, arbuscule-specific overexpression of MtSWEET1bY57A/G58D , which are considered to act in a dominant-negative manner, resulted in enhanced collapse of arbuscules. Taken together, our results reveal a (redundant) role for MtSWEET1b in the transport of glucose across the peri-arbuscular membrane to maintain arbuscules for a healthy mutually beneficial symbiosis.

Abstract 673: The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of this type of cancer is the loss of intercellular adhesion which is facilitated by inactivating mutations in E-cadherin. However the loss of E-cadherin alone is not enough to induce ILC. An in vivo Sleeping Beauty insertional mutagenesis screen was performed to identify genes that together with E-cadherin loss contribute to ILC development in mice. In around 85% of the tumors that were analyzed during this screen one of four hits were observed, namely MYPT1, MYPT2, ASPP2 and MYH9. Interestingly these four hits appear to be mutually exclusive indicating a shared mechanism of action. To investigate how these genes might affect tumorigenesis we first looked at the location of the transposon insertions. This revealed that for MYPT1, MYPT2 and ASPP2 the transposons appeared to localize to a specific region in the gene indicating that these insertions could result in a truncation variant. In the case of MYH9 no clear localization of insertions was found which in combination with the recently published data indicates a loss of function. Northern blot analysis revealed the presence of truncation variants for MYPT1/2 and ASPP2 which for MYPT1 were confirmed to also result in truncated protein variants. In order to identify the tumorigenic potential of truncated MYPT1 and ASPP2 we overexpressed them in spontaneously immortalized mammary epithelial cell lines HC11 and NMuMG. We are using these cell lines to analyse differences in cell proliferation, anchorage independent growth, sensitivity to apoptosis in vitro and tumor formation in vivo. To further analyse the hits in an E-cadherin negative setting we made use of primary mammary epithelial cells (MECs) isolated from genetically engineered mice with mammary gland specific E-cadherin loss. These MECs normally do not grow past several passages in vitro. However, these MECs when we overexpress truncated MYPT1 or ASPP2, or use shRNA mediated knockdown of MYH9 can be passaged and grown in vitro consistently. Finally we are generating conditional mouse models with mammary gland specific E-cadherin loss in combination of expression of the truncated variants of MYPT1 or ASPP2, or loss of MYH9. We will to monitor whether these mice are prone to developing ILC. Citation Format: Koen Schipper, Julian de Ruiter, Sjors Kas, Eva Schut, Marco Koudijs, David Addams, Jos Jonkers. The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 673.

SSTAR, a Stand-Alone Easy-To-Use Antimicrobial Resistance Gene Predictor.

We present the easy-to-use Sequence Search Tool for Antimicrobial Resistance, SSTAR. It combines a locally executed BLASTN search against a customizable database with an intuitive graphical user interface for identifying antimicrobial resistance (AR) genes from genomic data. Although the database is initially populated from a public repository of acquired resistance determinants (i.e., ARG-ANNOT), it can be customized for particular pathogen groups and resistance mechanisms. For instance, outer membrane porin sequences associated with carbapenem resistance phenotypes can be added, and known intrinsic mechanisms can be included. Unique about this tool is the ability to easily detect putative new alleles and truncated versions of existing AR genes. Variants and potential new alleles are brought to the attention of the user for further investigation. For instance, SSTAR is able to identify modified or truncated versions of porins, which may be of great importance in carbapenemase-negative carbapenem-resistant Enterobacteriaceae. SSTAR is written in Java and is therefore platform independent and compatible with both Windows and Unix operating systems. SSTAR and its manual, which includes a simple installation guide, are freely available from https://github.com/tomdeman-bio/Sequence-Search-Tool-for-Antimicrobial-Resistance-SSTAR-. IMPORTANCE Whole-genome sequencing (WGS) is quickly becoming a routine method for identifying genes associated with antimicrobial resistance (AR). However, for many microbiologists, the use and analysis of WGS data present a substantial challenge. We developed SSTAR, software with a graphical user interface that enables the identification of known AR genes from WGS and has the unique capacity to easily detect new variants of known AR genes, including truncated protein variants. Current software solutions do not notify the user when genes are truncated and, therefore, likely nonfunctional, which makes phenotype predictions less accurate. SSTAR users can apply any AR database of interest as a reference comparator and can manually add genes that impact resistance, even if such genes are not resistance determinants per se (e.g., porins and efflux pumps).

Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4+T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4+T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

Human genomics. The human transcriptome across tissues and individuals.

Transcriptional regulation and posttranscriptional processing underlie many cellular and organismal phenotypes. We used RNA sequence data generated by Genotype-Tissue Expression (GTEx) project to investigate the patterns of transcriptome variation across individuals and tissues. Tissues exhibit characteristic transcriptional signatures that show stability in postmortem samples. These signatures are dominated by a relatively small number of genes—which is most clearly seen in blood—though few are exclusive to a particular tissue and vary more across tissues than individuals. Genes exhibiting high interindividual expression variation include disease candidates associated with sex, ethnicity, and age. Primary transcription is the major driver of cellular specificity, with splicing playing mostly a complementary role; except for the brain, which exhibits a more divergent splicing program. Variation in splicing, despite its stochasticity, may play in contrast a comparatively greater role in defining individual phenotypes.

Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans.

Understanding the functional consequences of genetic variation, and how it affects complex human disease and quantitative traits, remains a critical challenge for biomedicine. We present an analysis of RNA sequencing data from 1641 samples across 43 tissues from 175 individuals, generated as part of the pilot phase of the Genotype-Tissue Expression (GTEx) project. We describe the landscape of gene expression across tissues, catalog thousands of tissue-specific and shared regulatory expression quantitative trait loci (eQTL) variants, describe complex network relationships, and identify signals from genome-wide association studies explained by eQTLs. These findings provide a systematic understanding of the cellular and biological consequences of human genetic variation and of the heterogeneity of such effects among a diverse set of human tissues.

Expression, genetic variation, and tissues

Human Genomics Human genomes show extensive genetic variation across individuals, but we have only just started documenting the effects of this variation on the regulation of gene expression. Furthermore, only a few tissues have been examined per genetic variant. In order to examine how genetic expression varies among tissues within individuals, the Genotype-Tissue Expression (GTEx) Consortium collected 1641 postmortem samples covering 54 body sites from 175 individuals. They identified quantitative genetic traits that affect gene expression and determined which of these exhibit tissue-specific expression patterns. Mele et al. measured how transcription varies among tissues, and Rivas et al. looked at how truncated protein variants affect expression across tissues. Science , this issue p. [648][1], p. [660][2], p. [666][3]; see also p. [640][4] [1]: /lookup/doi/10.1126/science.1262110 [2]: /lookup/doi/10.1126/science.aaa0355 [3]: /lookup/doi/10.1126/science.1261877 [4]: /lookup/doi/10.1126/science.aab3002

A novel mutation in the XPA gene results in two truncated protein variants and leads to a severe XP/neurological symptoms phenotype.

The nucleotide excision repair (NER) pathway repairs UV-induced DNA lesions in an accurate fashion and prevents UV-irradiated areas of the skin from tumour formation. The XPA protein plays a major role in DNA damage demarcation as well as stabilization of other NER factors and was found to be defective in xeroderma pigmentosum (XP) complementation group A patients. Characterization of four new XP-A patients. Genomic and cDNA sequencing, post-UV cell survival of living cells, host-cell reactivation of patients' fibroblasts and Western blotting. One of the four investigated patients shows a novel mutation leading to two different truncated protein variants. Three patients contain the already described p.R228X mutation. All patient cell lines exhibit a strong UVC sensitivity and reduced NER capability. In most of the cases stable protein expression was detected. We discovered four new XP-A patients and a novel XPA mutation resulting in two diverse patient alleles.

An eEF1A1 truncation encoded by PTI-1 exerts its oncogenic effect inside the nucleus

BackgroundThe oncogene PTI-1 was originally isolated from a prostate cancer cell line by its capability to transform rat fibroblasts. The PTI-1 mRNA has a very eccentric structure as the 5′UTR is similar to prokaryotic 23S rRNA, while the major open reading frame and the 3′UTR corresponds to a part of the mRNA encoding human translation elongation factor eEF1A1. Thus, the largest open reading frame encodes a truncated version of eEF1A1 lacking the first 67 amino acids, while having three unique N-terminal amino acids. Previously, the UTRs were shown to be a prerequisite for the transforming capacity of the PTI-1 transcript. In this study, we have investigated the possible role of the UTRs in regulating protein expression and localization.MethodsThe protein expression profiles of a number of PTI-1 mRNA variants were studied in vitro and in vivo. Furthermore, the oncogenic potentials of the same PTI-1 mRNAs were determined by monitoring the capacities of stably transfected cells expressing these mRNAs to induce tumors in nude mice and form foci in cell culture. Finally, the cellular localizations of PTI-1 proteins expressed from these mRNAs were determined by fluorescence microscopy.ResultsThe PTI-1 mRNA was found to give rise to multiple protein products that potentially originate from translation initiation at downstream, inframe AUGs within the major open reading frame. At least one of the truncated protein variants was also found to be oncogenic. However, the UTRs did not appear to influence the amount and identities of these truncated protein products. In contrast, our localization studies showed that the UTRs of the transcript promote a nuclear localization of the encoded protein(s).ConclusionsTranslation of the PTI-1 mRNA results in multiple protein products of which (a) truncated variant(s) may play a predominant role during cellular transformation. The PTI-1 UTRs did not seem to play a role in translation regulation, but appeared to contribute to a nuclear localization of the PTI-1 protein(s). This indicates that the PTI-1 protein(s) exert(s) its/their oncogenic function inside the nucleus.

ROR1 Isoforms Are Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) - a Survival Factor for CLL Cells

Abstract 1778 Background:Phosphorylation of receptor tyrosine kinases (RTK) plays an important role for many aspects of cell life, as cell-cycle progression, differentiation, apoptosis, intercellular communication and cell survival. RTKs as e.g. EGFR, HER2/neu, VEGFR, IGFR etc. are important structures contributing to the malignant phenotype of many tumors. Targeting RTKs by monoclonal antibodies (MAb) or small inhibitory molecules has been successful for the treatment of various cancers. ROR is one of the twenty RTK families and consists of two members, ROR1 and ROR2. ROR1 and ROR2 have important functions during embryogenesis. ROR1 is uniquely expressed in CLL cells compared to normal tissues. ROR1 siRNA transfection of CLL cells induced specific apoptosis of the leukemic cells. Aims:To study ROR1 protein variants in CLL, ROR1 phosphorylation and relation to clinical activity of the disease. Methods:Phosphorylation of primary human CLL cells from patients with non-progressive and progressive disease, and a series of cell lines were studied applying immunoprecipitation (IP) of the ROR1 molecule, using anti ROR1 and irrelevant MAbs by Western blotting as well as in intracytoplasmic staining of the cells by flowcytometry, using an anti-phospho ROR1 (pROR1) MAb (a MAb specifically recognizing a phospho-peptide of the cytoplasmic TK domain of ROR1) and semi quantification of the staining intensity as well as by p-tyrosine and p-serine MAbs. Results:IP of the ROR1 molecule of fresh leukemic cells using ROR1 specific antibodies and subsequent Western blot with anti ROR1 MAbs, showed various protein bands. Two bands had the size of 105 and 130 KDa probably representing unglycosylated or partially glycosylated ROR1 and the fully glycosylated glycoform respectively. One of the two bands dominated in individual patients. A band of 260 KDa could also be detected in a large number of patients probably representing dimerized monomers. Finally a band of 64 KDa could also be noted which may represent an intracellular part of ROR1, as has been described in fetal and adult human CNS, leukemia and lymphoma cell lines (Reddy UR et al, Oncogene. 1996; 13:1555–9). The 64, 105 and 130 KDa bands were constitutively phosphorylated in all patients both at tyrosine and serine residues. The intensity of phosphorylation of the 130 KDa band was significantly higher in patients with progressive disease vs. non-progressive disease (p=0.0001). There was no relation between the pattern of the different ROR1 protein bands and disease activity.Using 9 cell lines of different hematologic malignancies including CLL, similar protein bands and phosphorylation status as for fresh CLL cells were noted.We have also produced specific mouse monoclonal antibodies against defined epitopes of the extracellular parts of ROR1. One of those MAbs (anti-KNG, IgG1) induced a high degree of specific apoptosis of the CLL cells (ASH Annual Meeting Abstracts, Nov 2010; 116: 916) and could also be shown to induce a specific dephosphorylation of the cytoplasmic TK domain which was noted already after 20 min and gradually increased up to 4h. No effects were noted using irrelevant MAbs or healthy donor lymphocytes. Conclusion:Our results showed that the ROR1 molecule in CLL cells are expressed in various protein variants probably representing different glycosylation patterns (105–130 KDa), dimerized monomers (260 KDa) and a truncated protein variant (64 KDa). ROR1 was constitutively phosphorylated (64, 105 and 130 KDa) both at serine and tyrosine residues. Treatment of CLL cells in vitro with a ROR1 specific antibody induced specific apoptosis as well as rapid dephosphorylation of ROR1. Collectively the data suggest that phosphorylated ROR1 might be an important structure for the growth potential of CLL cells and an interesting structure to target in a therapeutic intervention. Disclosures:Mellstedt:Kancera AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hojjat-Farsangi:Kancera AB: Equity Ownership. Rabbani:Kancera AB: Equity Ownership.

Systematic Chromosomal Deletion of Bacterial Ribosomal Protein Genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection . Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA–protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.

Researchers Discover New Biomarker That May Improve Cancer Care Strategies

IN A PRELIMINARY STUDY, RESEARCHers identified a novel biomarker that predicts cancer metastasis. If validated in more-rigorous studies, the biomarker could help clinicians improve treatment strategies for patients with certain types of cancer. Researchers with the National Institutes of Health and the University of Hong Kong in China discovered that an N-terminal truncated protein variant of carboxypeptidase E (CPEN) induces tumor growth and metastasis. Patients with high levels of this protein were more likely to have their cancer spread, regardless of staging and grading; those with low levels, regardless of staging and grading, were more likely to not have their cancers spread (Lee TK et al. J Clin Invest. doi:10.1172 /JCI40433 [published online ahead of print February 1, 2011]). “We can tell from the levels [of CPEN] whether the tumor has spread and predict whether the tumor is likely to recur in the same tissue or spread to other parts of the body in the future,” said Y. Peng Loh, PhD, a coauthor of the research paper and a member of the National Institute of Child Health and Human Development’s section on cellular neurobiology. Because currently available biomarkers cannot accurately achieve such predictions, prognosis is determined by staging of the cancer using histopathological techniques, he noted. The findings are based on analysis of tissue from 99 patients with liver cancer, which involved a comparison of levels of CPEN RNA in the tumors and in surrounding tissue. Patients whose tumors had a CPEN RNA level that was more than twice that found in the surrounding tissue were highly likely to experience a recurrence or metastasis of their cancer within 2 years. Using this threshold measure, the researchers accurately predicted metastasis or recurrence in more than 90% of cases. Their predictions that tumors would not return in the 2-year period were accurate 76% of the time. Measuring CPEN levels was predictive regardless of the staging of the cancers. For example, in 18 patients with stage II cancer, a stage in which patients are often told their cancer is in remission and no further follow-up care is necessary, recurrence or metastasis occurred within 2 years in only 1 of 10 patients with low levels of CPEN and in 4 of 7 patients with high levels of CPEN. For 12 patients with stage IV liver cancer, 4 of 5 with low levels of CPEN did not experience recurrence 2 years postsurgery, while 6 of the 7 with high levels of CPEN did. “Ourassay,whichcanbetterdetermine theaggressivenessofthetumor,canbring hope of longer survival and psychologicalcomforttothosestageIVpatientswith low CPEN RNA levels,” Loh said. “It will also encourage practitioners to actively treat these patients with chemotherapy, since they have a good chance of not having a recurrence.” A biomarker that outperforms staging and grading after surgical resection could be clinically useful, said Stephen M. Hewitt, MD, PhD, another of the researchers and a staff scientist with the National Cancer Institute. “Tests such as this, where one can perform it on a small sample of the specimens without full resection, have the opportunity of allowing physicians to better plan patient care ahead of time and direct the care in a more effective manner,” Hewitt said. The researchers also studied 14 patientswithpheochromocytomaandparaganglioma, who were followed up for 8 years. For these patients, CPEN RNA levels (measured in the tumor tissue only because tissue surrounding the tumor was not obtainable), ranged from 150 000 to 15 million per 200 μg. In all cases in which cancer recurred or me-

CCN3 – A key regulator of the hematopoietic compartment

CCN3, a founding member of the CCN family of growth regulators, was linked with hematology in 2003(1) when it was detected in human serum. CCN3 is expressed and secreted by hematopoietic progenitor cells in normal bone marrow. CCN3 acts through the core stem cell signalling pathways including Notch and Bone Morphogenic Protein, connecting CCN3 with the modulation of self-renewal and maturation of a number of cell lineages including hematopoietic, osteogenic and chondrogenic. CCN3 expression is disrupted in Chronic Myeloid Leukemia as a consequence of the BCR-ABL oncogene and allows the leukemic clone to evade growth regulation. In contrast, naïve cord blood progenitors undergo enhanced clonal expansion in response to CCN3. Altered CCN3 expression is associated with numerous solid tumors including glioblastoma, melanoma, adrenocortical tumours, prostate cancer and bone malignancies including osteosarcoma. Mature CCN3 protein has five distinct modules and truncated protein variants with altered function are found in many cancers. Regulation by CCN3 is therefore cell type and isoform specific. CCN3 has emerged as a key player in stem cell regulation, hematopoiesis and a crucial component within the bone marrow microenvironment.