Rethinking Proteasome Evolution: Two Novel Bacterial Proteasomes

The proteasome is a multisubunit structure that degrades proteins. Protein degradation is an essential component of regulation because proteins can become misfolded, damaged, or unnecessary. Proteasomes and their homologues vary greatly in complexity: from HslV (heat shock locus v), which is encoded by 1 gene in bacteria, to the eukaryotic 20S proteasome, which is encoded by more than 14 genes. Despite this variation in complexity, all the proteasomes are composed of homologous subunits. We searched 238 complete bacterial genomes for structures related to the proteasome and found evidence of two novel groups of bacterial proteasomes. The first, which we name Anbu, is sparsely distributed among cyanobacteria and proteobacteria. We hypothesize that Anbu must be very ancient because of its distribution within the cyanobacteria, and that it has been lost in many more recent species. We also present evidence for a fourth type of bacterial proteasome found in a few β-proteobacteria, which we call β-proteobacteria proteasome homologue (BPH). Sequence and structural analyses show that Anbu and BPH are both distinct from known bacterial proteasomes but have homologous structures. Anbu is encoded by one gene, so we postulate a duplication of Anbu created the 20S proteasome. Anbu’s function appears to be related to transglutaminase activity, not the general stress response associated with HslV. We have found different combinations of Anbu, BPH, and HslV within these bacterial genomes, which raises questions about specialized protein degradation systems

A comparative reference study for the validation of HLA-matching algorithms in the search for allogeneic hematopoietic stem cell donors and cord blood units

The accuracy of human leukocyte antigen (HLA)-matching algorithms is a prerequisite for the correct and efficient identification of optimal unrelated donors for patients requiring hematopoietic stem cell transplantation. The goal of this World Marrow Donor Association study was to validate established matching algorithms from different international donor registries by challenging them with simulated input data and subsequently comparing the output. This experiment addressed three specific aspects of HLA matching using different data sets for tasks of increasing complexity. The first two tasks targeted the traditional matching approach identifying discrepancies between patient and donor HLA genotypes by counting antigen and allele differences. Contemporary matching procedures predicting the probability for HLA identity using haplotype frequencies were addressed by the third task. In each task, the identified disparities between the results of the participating computer programs were analyzed, classified and quantified. This study led to a deep understanding of the algorithms participating and finally produced virtually identical results. The unresolved discrepancies total to less than 1%, 4% and 2% for the three tasks and are mostly because of individual decisions in the design of the programs. Based on these findings, reference results for the three input data sets were compiled that can be used to validate future matching algorithms and thus improve the quality of the global donor search process

Crystal Structure of Outer Membrane Protein NMB0315 from Neisseria meningitidis

NMB0315 is an outer membrane protein of Neisseria meningitidis serogroup B (NMB) and a potential candidate for a broad-spectrum vaccine against meningococcal disease. The crystal structure of NMB0315 was solved by single-wavelength anomalous dispersion (SAD) at a resolution of 2.4 Å and revealed to be a lysostaphin-type peptidase of the M23 metallopeptidase family. The overall structure consists of three well-separated domains and has no similarity to any previously published structure. However, only the topology of the carboxyl-terminal domain is highly conserved among members of this family, and this domain is a zinc-dependent catalytic unit. The amino-terminal domain of the structure blocks the substrate binding pocket in the carboxyl-terminal domain, indicating that the wild-type full-length protein is in an inactive conformational state. Our studies improve the understanding of the catalytic mechanism of M23 metallopeptidases

Chromothripsis in acute myeloid leukemia: Biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix\uae) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology

Tumour Cell Heterogeneity.

The population of cells that make up a cancer are manifestly heterogeneous at the genetic, epigenetic, and phenotypic levels. In this mini-review, we summarise the extent of intra-tumour heterogeneity (ITH) across human malignancies, review the mechanisms that are responsible for generating and maintaining ITH, and discuss the ramifications and opportunities that ITH presents for cancer prognostication and treatment

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.We thank Prof. Miguel Lafarga for helpful comments and advice. We thank Dr Jose E Gomez-Arozamena for helping us with the irradiation experiments. We are grateful to Montse Fernandez Calderon, Susana Dawalibi, and Sonia Perez Mantecon, for excellent technical assistance. This work was supported by a Grant (BFU2017–84046-P) from the Spanish Science and Innovation Ministry to JAM. C.S.F is recipient of a FPI grant (BES-2015–074267)

Systemic IL-12 Administration Alters Hepatic Dendritic Cell Stimulation Capabilities

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2–3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support

Breaking point: the genesis and impact of structural variation in tumours

Somatic structural variants undoubtedly play important roles in driving tumourigenesis. This is evident despite the substantial technical challenges that remain in accurately detecting structural variants and their breakpoints in tumours and in spite of our incomplete understanding of the impact of structural variants on cellular function. Developments in these areas of research contribute to the ongoing discovery of structural variation with a clear impact on the evolution of the tumour and on the clinical importance to the patient. Recent large whole genome sequencing studies have reinforced our impression of each tumour as a unique combination of mutations but paradoxically have also discovered similar genome-wide patterns of single-nucleotide and structural variation between tumours. Statistical methods have been developed to deconvolute mutation patterns, or signatures, that recur across samples, providing information about the mutagens and repair processes that may be active in a given tumour. These signatures can guide treatment by, for example, highlighting vulnerabilities in a particular tumour to a particular chemotherapy. Thus, although the complete reconstruction of the full evolutionary trajectory of a tumour genome remains currently out of reach, valuable data are already emerging to improve the treatment of cancer