Structural analysis of the Sulfolobus solfataricus MCM protein N-terminal domain†

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 Å crystal structure of the N-terminal domain (residues 1–268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed

Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling.

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2–286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action

A CDC6-like factor from the archaea Sulfolobus solfataricus promotes binding of the mini-chromosome maintenance complex to DNA

The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms

X-Linked Parkinsonism: phenotypic and genetic heterogeneity

X-linked parkinsonism encompasses rare heterogeneous disorders mainly inherited as a recessive trait, therefore being more prevalent in males. Recent developments have revealed a complex underlying panorama, including a spectrum of disorders in which parkinsonism is variably associated with additional neurological and non-neurological signs. In particular, a childhood-onset encephalopathy with epilepsy and/or cognitive disability is the most common feature. Their genetic basis is also heterogeneous, with many causative genes and different mutation types ranging from "classical" coding variants to intronic repeat expansions. In this review, we provide an updated overview of the phenotypic and genetic spectrum of the most relevant X-linked parkinsonian syndromes, namely X-linked dystonia-parkinsonism (XDP, Lubag disease), fragile X-associated tremor/ataxia syndrome (FXTAS), beta-propeller protein-associated neurodegeneration (BPAN, NBIA/PARK-WDR45), Fabry disease, Waisman syndrome, methyl CpG-binding protein 2 (MeCP2) spectrum disorder, phosphoglycerate kinase-1 deficiency syndrome (PGK1) and X-linked parkinsonism and spasticity (XPDS). All clinical and radiological features reported in the literature have been reviewed. Epilepsy occasionally represents the symptom of onset, predating parkinsonism even by a few years; action tremor is another common feature along with akinetic-rigid parkinsonism. A focus on the genetic background and its pathophysiological implications is provided. The pathogenesis of these disorders ranges from well-defined metabolic alterations (PGK1) to non-specific lysosomal dysfunctions (XPDS) and vesicular trafficking alterations (Waisman syndrome). However, in other cases it still remains poorly defined. Recognition of the phenotypic and genetic heterogeneity of X-linked parkinsonism has important implications for diagnosis, management, and genetic counseling. \ua9 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Biochemical characterization of a CDC6-like protein from the crenarchaeon sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function

Lipid-rich histology in a basal-type immuno-profile breast carcinoma: a clinicopathological histochemical and immunohistochemical analysis of a case

We describe the clinicopathological and morphological features of an unusual breast carcinoma classifiable as a lipid-rich variant of ductal invasive carcinoma, with a basal-type immunohistochemical profile. Basal-type breast cancers show no hormonal receptor expression, rarely over-express HER-2 but exhibit molecular high weight cytokeratins, EGFR and c-kit positivity. Special stains and histochemistry tests were used to elucidate the nature of vescicles in the neoplastic cells. Sudan IV was performed on formalin-fixed tissue. Commercially available antibodies tested were: ER, PgR, EGFR, HER2, c-kit, high molecular weight cytokeratins. Cytoplasmic lipids were highlighted as red-orange droplets on Sudan IV staining. As for immunohistochemistry, the tumor showed no reactivity to ER, PgR and HER2 (triple negative), and diffuse and strong positivity to high weight cytokeratins, EGFR and c-kit, such as a basal-type breast carcinoma. A basaloid phenotype in a lipid-rich carcinoma has not been previously reported

Resource-aware construct design in mammalian cells

Resource competition can be the cause of unintended coupling between co-expressed genetic constructs. Here we report the quantification of the resource load imposed by different mammalian genetic components and identify construct designs with increased performance and reduced resource footprint. We use these to generate improved synthetic circuits and optimise the co-expression of transfected cassettes, shedding light on how this can be useful for bioproduction and biotherapeutic applications. This work provides the scientific community with a framework to consider resource demand when designing mammalian constructs to achieve robust and optimised gene expression

Carbohydrate Intake in the Etiology of Crohn's Disease and Ulcerative Colitis

Background: Diet may have a role in the etiology of inflammatory bowel disease. In previous studies, the associations between increased intakes of carbohydrates, sugar, starch, and inflammatory bowel disease are inconsistent. However, few prospective studies have investigated the associations between these macronutrients and incident Crohn's disease (CD) or ulcerative colitis (UC). Methods: A total of 401,326 men and women were recruited between 1991 and 1998. At recruitment, dietary intakes of carbohydrate, sugar, and starch were measured using validated food frequency questionnaires. The cohort was monitored identifying participants who developed incident CD or UC. Cases were matched with 4 controls, and odds ratios were calculated for quintiles of total carbohydrate, sugar, and starch intakes adjusted for total energy intake, body mass index, and smoking. Results: One hundred ten participants developed CD, and 244 participants developed UC during follow-up. The adjusted odds ratio for the highest versus the lowest quintiles of total carbohydrate intake for CD was 0.87, 95% CI = 0.24 to 3.12 and for UC 1.46, 95% CI = 0.62 to 3.46, with no significant trends across quintiles for either (CD, Ptrend = 0.70; UC, Ptrend = 0.41). Similarly, no associations were observed with intakes of total sugar (CD, Ptrend = 0.50; UC, Ptrend = 0.71) or starch (CD, Ptrend = 0.69; UC, Ptrend = 0.17). Conclusions: The lack of associations with these nutrients is in agreement with many case–control studies that have not identified associations with CD or UC. As there is biological plausibility for how specific carbohydrates could have an etiological role in inflammatory bowel disease, future epidemiological work should assess individual carbohydrates, although there does not seem to be a macronutrient effect

Warsaw Breakage Syndrome associated DDX11 helicase resolves G-quadruplex structures to support sister chromatid cohesion

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DD

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor