Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis

Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia. This mutation causes a late-stage 60S subunit maturation failure that targets mutant ribosomes for degradation. The resulting deficit in ribosomes causes the hypoproliferative phenotype. This 60S subunit shortage, in turn, exerts pressure on cells to select for suppressors of the ribosome biogenesis defect, allowing them to reestablish normal levels of ribosome production and cell proliferation. However, suppression at this step releases structurally and functionally defective ribosomes into the translationally active pool, and the translational fidelity defects of these mutants culminate in destabilization of selected mRNAs and shortened telomeres. We suggest that in exchange for resolving their short-term ribosome deficits through compensatory trans-acting suppressors, cells are penalized in the long term by changes in gene expression that ultimately undermine cellular homeostasis.Ribosomopathies are a family of congenital diseases that are linked to genetic defects in ribosomal proteins or ribosome biogenesis factors. They are characterized by pleiotropic abnormalities that include birth defects, heart and lung diseases, connective tissue disorders, anemia, ataxia, and mental retardation (reviewed in ref. 1). Although each ribosomopathy presents a unique pathological spectrum, the inherited forms are characterized by bone marrow failure and anemia early in life, followed by elevated cancer risk by middle age. For example, although childhood anemia is one of the cardinal symptoms of the genetically inherited disease Diamond–Blackfan anemia, these patients have a fivefold higher lifetime risk of cancer than the general population and a 30- to 40-fold higher risk of developing acute myeloid leukemia, osteosarcoma, or colon cancer (reviewed in refs. 2, 3). Similarly, patients with X-linked dyskeratosis are predisposed to myeloid leukemia and a variety of solid tumors (4), whereas patients with 5q− syndrome are at higher risk of developing acute myeloid leukemia (reviewed in ref. 5). In the genetically tractable zebrafish model, heterozygous loss-of-function mutations in several ribosomal proteins cause development of peripheral nerve sheet tumors (6). Somatically acquired mutations in ribosomal proteins are also implicated in cancer: ∼10% of children with T-cell acute lymphoblastic leukemia (T-ALL) were found to harbor somatic mutations in the ribosomal protein of the large subunit (LSU) 10, 5, and 22 (RPL10, RPL5, and RPL22) (7). [Note that the proteins encoded by these genes are also named uL16, uL18, and eL22, respectively, under the newly proposed uniform ribosomal protein nomenclature (8).] A separate study identified heterozygous deletions in the region of chromosome 1p that contains RPL22 (eL22) in an additional 10% of patients with T-ALL (9). The model of ribosomal proteins as targets for somatic mutations in cancer is further supported by the finding that two ribosomal protein genes (RPL5/uL18 and RPL22/eL22) are included in the list of 127 genes identified as significantly mutated in cancer in the context of the first Cancer Genome Atlas pan-cancer analysis in 12 tumor types (10).Ribosomopathies present an intriguing paradox: Although patients initially present with hypoproliferative disorders (e.g., anemias, bone marrow failure), those who survive to middle age often develop hyperproliferative diseases (i.e., cancers). The link between ribosome defects and hypoproliferative disease phenotypes has been extensively studied: The current working hypothesis is that impaired ribosome biogenesis activates a “ribosomal stress” cascade, activating the cellular TP53 pathway and resulting in cell cycle arrest and cell death (11). However, activation of TP53 does not explain why ribosomal defects are associated with hyperproliferative diseases, particularly cancer. Mutations in the ribosomal protein gene RPL10/uL16 were recently identified in patients with T-ALL (7). The T-ALL–associated RPL10/uL16 mutations occurred almost exclusively in residue arginine 98 (R98), with the exception of one patient harboring the Q123P mutation, which lies adjacent to R98 within the rpL10/uL16 3D structure (Fig. 1). Both residues are at the base of an essential flexible loop in rpL10 that closely approaches the peptidyltransferase center in the catalytic core in the ribosome (12). In addition to its role in catalysis (13, 14), rpL10/uL16 plays an important role in the late stages of 60S subunit biogenesis. After initial production of the separate ribosomal subunits in the nucleus, immature and functionally inactive pre-60S subunits are exported to the cytoplasm, where they undergo additional maturation events (15), including incorporation of rpL10/uL16, before they can associate with mature 40S subunits and engage in protein synthesis (16). Among the critical set of final 60S maturation steps is the release of the antiassociation factor Tif6, followed by release of Nmd3, the primary export adaptor for the pre-60S subunit in yeast and in humans (17, 18). Tif6 release requires the tRNA structural mimic Sdo1p (19) and the GTPase Efl1, a paralog of eukaryotic elongation factor 2 (eEF2) (20). We have suggested that structural rearrangements of the internal loop of rpL10/uL16 coordinate this final maturation process, resulting in a test drive of the pre-60S subunit to ensure that only properly functioning subunits are allowed to enter the pool of translationally active ribosomes (13, 21). Defective ribosomes carrying mutations in rpL10/uL16 specifically fail in this test drive, leading to their degradation through a molecular pathway that is yet to be characterized. Beyond 60S maturation, rpL10/uL16 plays an important role in coordinating intersubunit rotation and controlling allosteric rearrangements within the ribosome, helping to ensure the directionality and fidelity of protein synthesis (13).Open in a separate windowFig. 1.Localization of rpL10 and the loop in the LSU. (A) rpL10/uL16 in the context of the crown view of the LSU. (B) Close-up view of rpL10/uL16 and the local environment. The flexible loop structure is indicated by dashed red lines, and the positions of R98 and Q123 are indicated. rpL10/uL16 is situated between helices 38 and 89, and it is located in close proximity to several functional centers of the LSU, including the peptidyltransferase center (PTC), aa-tRNA accommodation corridor, and elongation factor binding site. Images were generated using PyMOL.rpL10/uL16 is highly conserved among eukaryotes: The yeast and human proteins are interchangeable, and residue 98 is invariantly an arginine (22). Human RPL10/uL16 is located on the X chromosome, and is therefore expressed as a single-copy gene in males. Thus, the haploid yeast model is an excellent mimic of the situation in the cells of a patient with T-ALL. Yeast cells expressing rpl10-R98S, rpl10-R98C, and rpl10-H123P (corresponding to Q123 in human rpL10/uL16) as the sole forms of rpL10/uL16 displayed proliferative defects. Further, polysome profiling revealed increased ratios of free 60S and 40S subunits vs. monosomes, markedly reduced polysomes, and the presence of halfmers in these mutants, suggesting defects in both ribosome biogenesis and subunit joining (7). Tif6 and Nmd3 both accumulated in the cytoplasm in the mutant cells, indicating a defect in their release from the cytoplasmic 60S (7). Thus, all of the rpl10/uL16 mutations appeared to affect 60S biogenesis at the Efl1-dependent quality control step. Consistent with the yeast-based observations, mouse lymphoid cells expressing rpl10-R98S displayed slower proliferation rates than cells expressing WT RPL10/uL16 and conferred defective polysome profiles (7).The studies presented in the current report use the yeast rpl10-R98S mutant to elucidate the structural, biochemical, and trans-lational fidelity defects that may lead to carcinogenesis. This mutant perturbs the structural equilibrium of ribosomes toward the “rotated state.” At the biochemical level, this underlying structural defect alters the affinity of mutant ribosomes for a specific set of trans-acting ligands. In turn, the biochemical defects affect translational fidelity, promoting elevated rates of −1 programmed ribosomal frameshifting (−1 PRF) and impaired recognition of termination codons. Globally increased rates of −1 PRF result in a decreased abundance of cellular mRNAs that harbor operational −1 PRF signals (23, 24). These −1 PRF signal-containing mRNAs include EST1, EST2, STN1, and CDC13, which play central roles in yeast telomere maintenance (23). In rpl10-R98S cells, the steady-state abundances of these mRNAs are decreased, resulting in telomere shortening. A spontaneously acquired trans-acting mutant suppresses the ribosome biogenesis defects of the rpl10-R98S mutant, thereby reestablishing high levels of ribosome production and cell proliferation. Importantly, however, suppression of the biogenesis and growth impairment defects fails to suppress the profound structural, biochemical, and translational fidelity defects of rpL10-R98S ribosomes. These findings suggest that suppression of the growth defect results from bypassing the test drive. Although the suppressor mutation enables cells to grow at normal rates, genetic suppression comes at the cost of releasing functionally defective ribosomes into the translationally active pool. We propose two different but not mutually exclusive models for how somatically acquired rpL10/uL16 mutations may promote cancer: (i) Mutant ribosomes may drive altered gene expression programs, promoting T-ALL, or (ii) the suppressor mutations may themselves be the drivers of T-ALL.     

Differential global structural changes in the core particle of yeast and mouse proteasome induced by ligand binding

Two clusters of configurations of the main proteolytic subunit β5 were identified by principal component analysis of crystal structures of the yeast proteasome core particle (yCP). The apo-cluster encompasses unliganded species and complexes with nonpeptidic ligands, and the pep-cluster comprises complexes with peptidic ligands. The murine constitutive CP structures conform to the yeast system, with the apo-form settled in the apo-cluster and the PR-957 (a peptidic ligand) complex in the pep-cluster. In striking contrast, the murine immune CP classifies into the pep-cluster in both the apo and the PR-957–liganded species. The two clusters differ essentially by multiple small structural changes and a domain motion enabling enclosure of the peptidic ligand and formation of specific hydrogen bonds in the pep-cluster. The immune CP species is in optimal peptide binding configuration also in its apo form. This favors productive ligand binding and may help to explain the generally increased functional activity of the immunoproteasome. Molecular dynamics simulations of the representative murine species are consistent with the experimentally observed configurations. A comparison of all 28 subunits of the unliganded species with the peptidic liganded forms demonstrates a greatly enhanced plasticity of β5 and suggests specific signaling pathways to other subunits.Among the many factors involved in protein degradation through the ubiquitin-proteasome pathway, the core particle (CP) 20S proteasome plays the key role of the protease component. With the regulatory particle (RP), it forms a complex that selectively degrades ubiquitin-protein conjugates (1, 2). The CP in eukaryotes is a multisubunit complex composed of four stacked heptameric rings: two identical outer rings formed by seven different α subunits and two identical inner rings formed by seven different β subunits. The α_1–7β_1–7β_1–7α_1–7 organization defines a cylindrical structure (3). The α-rings control substrate entry into the lumen of the particle, where it is processed at the peptidolytic active centers, which are located at the inner walls of the β rings, specifically at subunits β1, β2, and β5. These active subunits are characterized by an N-terminal Thr residue. The other four β subunits have unprocessed N-terminal propeptides and are enzymatically inactive.All three active subunits share a common peptide hydrolyzing mechanism with two main steps (4): (i) the positioning of the substrate peptide in the active site by antiparallel alignment in between segments 47–49 and 21 of the active β subunits and (ii) peptide bond cleavage initiated by a nucleophilic attack of the hydroxyl group of the N-terminal Thr1 on the carbonyl carbon atom of the scissile peptide. Sequence diversity among β subunits endows them with distinctive structural features and different specificity pockets (S1, S2, S3, etc.) where the substrate side chains (P1, P2, P3, etc.) are bound (5). Consequently, the correlation of structural features of the S1 pockets with the distinctive cleavage products has led to the association of β1, β2, and β5 with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively (6).The catalytically active subunits are substituted in immune cells of vertebrate organisms by the immune β-subunits β1i, β2i, and β5i as part of an adaptive immune response. These substitutions cause substantial functional differences between the constitutive (cCP) and immuno (iCP) species, reflected in higher yield of peptides that are recognized by the major histocompatibility complex (MHC) class I generated by iCP (7). Additionally, it has been observed that iCP achieves higher degradation rates than cCP, in both in vitro and cellular assays (8–13).Some sequence variations between the constitutive and immune subunits provide explanations to the observed catalytic differences. Most conspicuously, and first seen in the eukaryotic proteasome crystal structure from yeast (yCP) (3) and confirmed by the murine constitutive and immune CP structures (mcCP and miCP) (14), Arg45 of the β1 subunit, located at the base of the S1 pocket, is replaced by leucine in β1i, thereby causing a specific change of the electrostatic milieu, in line with the observed low postacidic activity of the iCP (15).Despite the high sequence similarity between β5 subunits of mcCP and miCP including identical active sites, a peptidic α-β-epoxyketone inhibitor, PR-957, showed higher affinity to iCP by one order of magnitude. The structural comparison of cCP and iCP in their apo and PR-957 liganded states suggested an explanation. On binding of PR-957, the cCP β5 backbone displays significant deformations, whereas the iCP β5 backbone remains unchanged. This observation, together with our experience in constructing β5 models for virtual screening purposes, prompted us to reinvestigate the vast amount of structural data for yCP by a procedure that facilitates discovery of global changes: principal component analysis (PCA).We focus our study on the β5 subunit, because β5 inactivation in yeast renders a lethal phenotype (16) and therefore β5 harbors an essential enzymatic activity, and because almost all crystallographically defined complexes are liganded at their β5 active site.Here we present a detailed investigation of the wealth of yeast and mouse proteasome ligand complex structures that led us to embark on structural comparisons beyond the immediate vicinity of the ligands to obtain a view of the global response of the core particle of yeast and mouse proteasome to complex formation. This study (i) is evidence of the structural plasticity of the β, specifically β5, subunits; (ii) offers perspectives for the analysis of the structure-function relationship of the CP; and (iii) provides an aid for the design and development of ligands as drugs for this intensively studied target for cancer and autoimmune diseases.     

硒蛋白S基因G-254A位点多态性与食管癌发病风险的关联性

目的探讨内蒙古汉族人群硒蛋白(Sel)S基因G-254A位点多态性与食管癌发病风险的相关性。方法采用四引物扩增阻碍突变体系聚合酶链式反应检测124例内蒙古汉族食管癌患者和132例健康对照者的基因型和等位基因频率,并进行测序验证,分析两组SelS基因G-254A位点基因型和等位基因频率分布的差异,同时对食管癌发病风险进行分层分析和多因素Logistic回归分析。结果吸烟、饮酒和基因突变是食管癌发病的主要危险因素(OR分别为1.782、1.594、2.286,均P<0.1);SelS基因G-254A位点CT、CC、TT基因型和C、T等位基因频率在两组间存在统计学差异(均P<0.05)。内蒙古汉族人群中携带T等位基因的CT和(或)TT基因型者患食管癌的风险高于CC基因型者(OR=2.005,95%CI:1.36~2.591),携带T等位基因且吸烟的个体患食管癌的风险增加,是CC基因型的2.611倍(OR=2.611,95%CI:1.410~4.835)。结论吸烟、饮酒和基因突变是食管癌发病的主要危险因素,吸烟与SelS基因突变可交互作用增加食管癌发病风险。     

高通量测序在鸟类肠道微生物中的研究进展

鸟类作为分布广泛、种群数量庞大和高度多样化的物种之一,在病原体传播中发挥了重要作用。本文综述了基于高通量测序技术的3种测序策略(16S rDNA测序、宏基因组测序和宏转录组测序)的特点及其在鸟类肠道菌群多样性、抗生素抗性基因和病原微生物发现中的应用,并对未来发展趋势进行了展望。     

Failure of productive infection of Mallards (Anas platyrhynchos) with H16 subtype of avian influenza viruses

Background

Mallard ducks and other waterfowl represent the most important reservoirs of low pathogenic avian influenza viruses (LPAIV). In addition, mallards are the most abundant duck species in Eurasia that migrate over long distances. Despite extended wild bird monitoring studies over the past decade in many Eurasian countries and investigating hundreds of thousands of wild bird samples, no mallard duck was found to be positive for avian influenza virus of subtype H16 in faecal, cloacal or oropharyngeal samples. Just three cases of H16 infections in Anseriformes species were described worldwide. In contrast, H16 viruses have been repeatedly isolated from birds of the Laridae family.

Objective

Here, we tested the hypothesis that mallards are less permissive to infection with H16 viruses.

Methods

Groups of mallard ducks of different age were inoculated via the oculo-nasal-oral route with different infectious doses of an H16N3 AIV.

Results

The ducks did not show any clinical symptoms, and no virus shedding was evident from cloacal and respiratory routes after experimental infection as shown by negative RT-qPCR results. In addition, all serum samples taken on days 8, 21 and 24 post-inoculation were negative by competitive NP-ELISA.

Conclusions

This study provided evidence that mallards are resistant to infection with H16N3 LPAIV.     

Exploring the relationship between cognitive illness representations and poor emotional health and their combined association with diabetes self-care. A systematic review with meta-analysis

Objective

Depression and anxiety are common in diabetes and are associated with lower diabetes self-care adherence. How this occurs is unclear. Our systematic review explored the relationship between cognitive illness representations and poor emotional health and their combined association with diabetes self-care.

Methods

Medline, Psycinfo, EMBASE, and CINAHL were searched from inception to June 2013. Data on associations between cognitive illness representations, poor emotional health, and diabetes self-care were extracted. Random effects meta-analysis was used to test the relationship between cognitive illness representations and poor emotional health. Their combined effect on diabetes self-care was narratively evaluated.

Results

Nine cross-sectional studies were included. Increased timeline cyclical, consequences, and seriousness beliefs were associated with poorer emotional health symptoms. Lower perceived personal control was associated with increased depression and anxiety, but not mixed anxiety and depressive symptoms. Remaining cognitive illness representation domains had mixed statistically significant and non-significant relationships across emotional states or were measured only once. Effect sizes ranged from small to large (r = ± 0.20 to 0.51). Two studies explored the combined effects of cognitions and emotions on diabetes self-care. Both showed that cognitive illness representations have an independent effect on diabetes self-care, but only one study found that depression has an independent effect also.

Conclusions

Associations between cognitive illness representations and poor emotional health were in the expected direction — negative diabetes perceptions were associated with poorer emotional health. Few studies examined the relative effects of cognitions and emotions on diabetes self-care. Longitudinal studies are needed to clarify directional pathways.     

Tamoxifen induces resistance to activated protein C

Introduction

The estrogen antagonist tamoxifen (TAM) increases the thrombotic risk similar to estrogen containing oral contraceptives (OC). In OC users this risk is attributed to alterations of hemostasis resulting in acquired resistance to activated protein C (APC). TAM-induced APC resistance has not been reported yet.

Materials and Methods

Blood samples were collected prospectively from women with breast cancer before (n = 25) and monthly after start of adjuvant TAM treatment (n = 75). APC resistance was evaluated on basis of the effect of APC on the endogenous thrombin generation potential. To detect increased in vivo APC generation APC plasma levels were measured using a highly sensitive oligonucleotide-based enzyme capture assay. Routine hemostasis parameters were measured additionally.

Results

APC sensitivity decreased by 41% (p = 0.001) compared to baseline after one month of TAM application and remained significantly decreased during the study period. Free protein S increased (p = 0.008) while other analyzed procoagulant factors, inhibitors, and activation markers of coagulation decreased or did not change significantly. In five patients the APC concentration increased to non-physiological levels but an overall significant increase of APC was not observed.

Conclusions

This is the first study showing acquired APC resistance under TAM therapy. Acquired APC resistance might explain the increased thrombotic risk during TAM treatment. Observed changes of hemostasis parameters suggest different determinants of TAM-induced APC resistance than in OC-induced APC resistance. The presence of acquired APC resistance in TAM patients warrants further evaluation if these patients may benefit from antithrombotic prophylaxis in the presence of additional thrombotic risk factors.     

Risk factors and clinical profile of thrombotic thrombocytopenic purpura in systemic lupus erythematosus patients. Is this a distinctive clinical entity in the thrombotic microangiopathy spectrum?: A case control study

Introduction

The association of thrombotic thrombocytopenic purpura (TTP) with systemic lupus erythematosus (SLE) is rare. It is associated with high morbidity and mortality. Information about risk factors and clinical outcomes is scant.

Material and Methods

A retrospective case-control study was performed in a referral center in Mexico City between 1994 and 2013. Patients were diagnosed with TTP if they fulfilled the following criteria: microangiopathic haemolytic anaemia, thrombocytopenia, high LDH levels, normal fibrinogen and negative Coombs’ test. Patients with SLE were diagnosed with ≥ 4 ACR criteria. We included three study groups: group A included patients with SLE-associated TTP (TTP/SLE; cases n = 22, TTP events n = 24); patients with non-autoimmune TTP (NA-TTP; cases n = 19, TTP events n = 22) were included in group B and patients with SLE without TTP (n = 48) in group C.

Results

After multivariate analysis, lymphopenia < 1000/mm3 [OR 19.84, p = 0.037], high SLEDAI score three months prior to hospitalisation [OR 1.54, p = 0.028], Hg < 7 g/dL [OR 6.81, p = 0.026], low levels of indirect bilirubin [OR 0.51, p = 0.007], and less severe thrombocytopenia [OR 0.98, p = 0.009] were associated with TTP in SLE patients. Patients with TTP/SLE received increased cumulative steroid dose vs. NA-TTP (p = 0.006) and a higher number of immunosuppressive drugs (p = 0.015). Patients with TTP/SLE had higher survival than NA-TTP (p = 0.033); however, patients hospitalised for TTP/SLE had a higher risk of death than lupus patients hospitalised for other causes

Conclusions

Lymphopenia is an independent risk factor for TTP/SLE. It is likely that patients with TTP/SLE present with less evident clinical features, so the level of suspicion must be higher to avoid delay in treatment.     

Impacts of HIV infection and long-term use of antiretroviral therapy on the prevalence of oral human papilloma virus type 16

J Oral Pathol Med (2012) 41 : 309–314 Background: The objectives of this study were to determine (i) the prevalence and the copy numbers of oral human papilloma virus type 16 (HPV‐16) in HIV‐infected patients compared with non‐HIV controls, and (ii) the effects of antiretroviral therapy (ART) and its duration on the virus. Methods: A cross‐sectional study was carried out in HIV‐infected patients with and without ART and in non‐HIV controls. Saliva samples were collected, and the DNA extracted from those samples was used as a template to detect HPV‐16 E6 and E7 by quantitative polymerase chain reaction. Student’s t‐test and ANOVA test were performed to determine the prevalence rates among groups. Results: Forty‐nine HIV‐infected patients: 37 on ART (age range, 23–54 years; mean, 37 years), 12 not on ART (age range, 20–40 years; mean, 31 years), and 20 non‐HIV controls (age range, 19–53 years; mean, 31 years) were enrolled. The prevalence of oral HPV‐16 infection and the copy numbers of the virus were significantly higher in HIV‐infected patients than in non‐HIV controls when using E6 assay (geometric mean = 10696 vs. 563 copies/10⁵ cells, P < 0.001), but not E7 assay. No significant difference was observed between those who were and were not on ART. Long‐term use of ART did not significantly change the prevalence of oral HPV‐16 infection and the copy numbers of the virus (P = 0.567). Conclusion: We conclude that the prevalence of oral HPV‐16 infection and the copy numbers of the virus are increased by HIV infection. Neither the use of ART nor its duration significantly affected the virus.