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In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation., (© 2023. The Author(s).)

Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein transport into the nucleus is mediated by transport receptors. Import of highly charged proteins, such as histone H1 and ribosomal proteins, requires a dimer of two transport receptors. In this study, we determined the cryo-EM structure of the Imp7:Impβ:H1.0 complex, showing that the two importins form a cradle that accommodates the linker histone. The H1.0 globular domain is bound to Impβ, whereas the acidic loops of Impβ and Imp7 chaperone the positively charged C-terminal tail. Although it remains disordered, the H1 tail serves as a zipper that closes and stabilizes the structure through transient non-specific interactions with importins. Moreover, we found that the GGxxF and FxFG motifs in the Imp7 C-terminal tail are essential for Imp7:Impβ dimerization and H1 import, resembling importin interaction with nucleoporins, which, in turn, promote complex disassembly. The architecture of many other complexes might be similarly defined by rapidly exchanging electrostatic interactions mediated by disordered regions., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1.0 overexpressed in Escherichia coli, as well as its domains: N-terminal/globular domain and C-terminal intrinsically disordered domain. This purification protocol relies on a simple affinity chromatography step followed by cation exchange due to the highly basic properties of histone proteins. Therefore, this protocol can also be applied to other linker histones. Highly pure proteins in amounts sufficient for most biochemical experiments can be obtained. The functional quality of purified H1.0 histone and its domains has been confirmed by pull-down, gel-mobility shift assays and the nuclear import assay.

Amino acid:[carrier protein] ligases (aa:CP ligases) are recently discovered enzymes that are highly similar to class II aminoacyl-tRNA synthetases (aaRSs). However, while aaRSs aminoacylate tRNA and supply building blocks for ribosomal translation, aa:CP ligases transfer activated amino acids to the phosphopantetheine group of small carrier proteins. We have solved the crystal structure of an aa:CP ligase complexed with the carrier protein (CP). The CP prosthetic group enters the active site from a different direction than tRNA in class II aaRS complexes through an idiosyncratic tunnel. CP binds to aa:CP ligase in a fundamentally different manner compared to tRNA binding by structurally closely related aaRSs. Based on crystallographic analysis, an enzyme of altered CP specificity was designed, and the mechanism of amino acid transfer to the prosthetic group was proposed. The presented study reveals how a conserved class II aaRS catalytic core can adapt to another function through minor structural alterations.

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.

Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRS-like proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.

All seryl-tRNA synthetases (SerRSs) are functional homodimers with a C-terminal active site domain typical for class II aminoacyl-tRNA synthetases and an N-terminal domain involved in tRNA binding. The recently solved three-dimensional structure of Methanosarcina barkeri SerRS revealed the idiosyncratic features of methanogenic-type SerRSs; that is, an active site zinc ion, a unique tRNA binding domain, and an insertion of approximately 30 residues in the catalytic domain, which adopt a helix-turn-helix (HTH) fold. Here, we present biochemical evidence for multiple roles of the HTH motif; it is important for dimerization of the enzyme, contributes to the overall stability, and is critical for the proper positioning of the tRNA binding domain relative to the catalytic domain. The changes in intrinsic fluorescence during denaturation of the wild-type M. barkeri SerRS and of the mutated variant lacking the HTH motif combined with cross-linking and gel analysis of protein subunits during various stages of the unfolding process revealed significantly reduced stability of the mutant dimers. In vitro kinetic analysis of enzymes, mutated in one of the N-terminal helices and the HTH motif, shows impaired tRNA binding and aminoacylation and emphasizes the importance of this domain for the overall architecture of the enzyme. The role of the idiosyncratic HTH motif in dimer stabilization and association between the catalytic and tRNA binding domain has been additionally confirmed by a yeast two-hybrid approach. Furthermore, we provide experimental evidence that tRNA binds across the dimer.

Coronary subclavian steal syndrome is a possible sequel in patients who have undergone myocardial revascularization with an internal mammary artery. We report a case of this syndrome in a 67-years-old man. In 1990 he underwent a quadruple bypass: aorta-obtuse margin, aorta-right coronary (two sequential), internal artery mammary-descending coronary artery. Three months later he started to have angina pectoris. In April 1992 an aortic arch angiography and a coronary angiography were performed. The examination showed an occlusion of the left subclavian artery at its origin. The artery was opacified countercurrently by the left vertebral artery but the left mammary artery was not opacified. Left coronary angiography showed a very severe disease of left anterior descending coronary artery and retrograde flow through the anastomosis in the left mammary artery. The patient underwent a left common carotid-subclavian artery bypass operation using a 6 mm vascutex graft. Eighteen months later the patient is doing well without angina pectoris and with very little alteration of the perfusion in the left frontal lobe observed by SPECT neuroimaging with a lipophilic tracer (99mTc-HMPAO). We think that the coronary-subclavian steal syndrome can be treated successfully with low risk by means of common carotid-subclavian artery bypass.

The incidence of chylothorax after pleuro-pulmonary operations as well as its treatment is reported. Of 1744 operations performed postoperative chylothorax developed in 13 (0.74%). It resulted in two cases from the transection of the thoracic duct, in six from the transection of the so-called minor lymph channels, which drain lymph from mediastinal nodes straight into the thoracic duct or drain lung segments into the thoracic duct via the pulmonary ligament; the transection of these channels occurred during mediastinal lymphadenectomy or during the section of the pulmonary ligament. In 5 patients the site of leakage was not determined as reoperation was not required. Conservative treatment with low-fat diet and medium-chain triglycerides and/or total parenteral nutrition was attempted in all but one patient but was successful only in 5 cases whose mean losses were 292 ml/day. Seven patients were reoperated after a mean of 11 days; their mean losses were 930 ml/day. One patient was reoperated on the third postoperative day without attempting conservative treatment; his mean loss was 850 ml/day. Lymphadenectomy seems to be an important risk factor for postoperative chylothorax. Chyle leakage around 500 ml/day or higher that tends to decrease below 500 ml/day after a few days of dietary manipulation usually stops within 10-20 days, while leakage over 500 ml/day that does not tend to decrease below 500 ml/day seldom if ever stops without surgery, so that a more aggressive attitude is justified.

From 1980 through 1993 ten patients underwent concomitant coronary artery bypass grafting and lung resection via median sternotomy. In eight patients a lung malignancy was resected, of which one was a small cell lung cancer. The lung resection was carried out before cardiopulmonary bypass in eight patients and during cardiopulmonary bypass in two. Coronary artery bypass grafting was performed using saphenous vein in eight patients; internal mammary artery was used as arterial conduit in two patients. There was one postoperative death while postoperative complications during hospital stay occurred in two patients. Pulmonary bleeding did not occur in any patient in whom lung resection was performed either before or during cardiopulmonary bypass. Both the patients who had internal mammary artery grafting experienced complications related to an associated lobectomy. A staged procedure is advisable if internal mammary artery has to be used and a lobectomy is required. The long-term survival in the patients with lung cancer was less than expected but the number of patients is too small to draw definite conclusions.

A 67-year-old man presenting with oppressive atypical chest pain 13 years after aorto-coronary saphenous vein bypass surgery, was found to have a large left anterior mediastinal mass on chest x-rays. Computerized tomography and digital subtraction angiography showed conflicting evidence on the vascular nature of the structure: the suspect diagnosis was of aortic pseudoaneurysm originating in the area of previous aortic cannulation. Selective coronary and bypass angiography showed extreme irregularity and dilatation of the grafts, and revealed the mediastinal mass to be represented by aneurysmal dilatation of the saphenous vein graft to the obtuse marginal coronary artery, partially filled with thrombus. At operation, another smaller aneurysm was found on the right coronary artery graft. Aneurysmal degeneration of vein grafts in the coronary circulation is exceedingly rare, and in only 6 reported cases has such an aneurysm presented as a mediastinal mass. To our knowledge, this is the first case in which an aneurysmal dilatation involved two saphenous vein coronary grafts.

We describe a case of left ventricular intramyocardial lipoma in a patient symptomatic for chest pain and syncope. The two-dimensional echocardiographic investigation showed a hyperechogenic mass inside the interventricular septum; presence of the mass was confirmed by CT scan and nuclear magnetic resonance imaging. In our experience only the CT scan was useful to clearly identify the histologic characteristics of tissue. The patient underwent successful surgical removal of the mass and the histologic examination revealed normal fat tissue.

Ventricular aneurysm is a frequent complication of myocardial infarction, but its diagnostic and prognostic implications are not fully defined. A surgical series of 66 ventricular resections for symptomatic left ventricular aneurysm is analyzed. Pts. age varied between 26-29 years; the interval between the episode of infarction and operation from 2 mos. to 12 years. Indication for surgery was angina in 53% of the cases, heart failure in 23%, arrhythmias in 15%, angina and failure in 9%. In 55 cases surgical findings were consistent with true aneurysm in the anatomical sense, in 11 cases with a wide area of wall akinesia. In 24 cases endocavitary thrombosis was present. In 23 cases aneurysmectomy was the only surgical procedure, with closure of acquired VSD in 1 case and mitral valve prosthesis in 2 cases. In 43 cases aorto-coronary bypass grafting was associated (single bypass in 18, double in 23, triple in 2). Operative technique underwent modifications in time, in relation to the use of local and general hypothermia, of cardioplegia, of early aortic cross-clamping. Surgical mortality was of 7.5% (5 cases); the actuarial courve, including operative mortality, showed survival of 88% at 1 year and of 78% at 5 years interval. Correlation between mortality and clinical symptomatology, hemodynamic data and surgical findings was not statistically significant; a statistically highly significant correlation was found between mortality and operative technique. The results are interpreted and discussed in relation to the diagnostic definition of the problem, to the criteria of indication to surgery and to the operative technique.

During the period 1966-1977, 705 pacemaker implantations with transvenous endocardial electrode and 369 substitutions of generators were carried out, besides 275 operations treating some complications after implantation were required. The most frequent complications were: endocardial electrode dislocation (9.4%), lesion caused by decubitus (7.5%) and infection of subcutaneous pocket (5%), decubitus lesion and infection of the skin caused by the electrode (1.7%), electrode and generator breaking (1.3%), right ventricle perforation caused by the electrode (0.37%). Surgical mortality-rate was 0.18%.

We describe the case of a patient with massive pulmonary embolism, in which two-dimensional echocardiography demonstrated the presence of a free-floating mass in the right atrial cavity. The patient was urgently and successfully operated on. We describe the echocardiographic findings and emphasize the value of two dimensional echocardiography in detecting right-sided intracavitary thromboemboli, a condition in which immediate surgical treatment is mandatory.

The case is presented by a 54 year-old woman, studied because of a clinical diagnosis of mitral valve disease, in which echocardiographic and angiocardiographic examination afforded the diagnosis of left atrial tumor. The surgical finding was of a large myxoma implanted on the atrial septum and prolapsing into the mitral opening and engaging with a branch into an atrial septal defect of the cavalis superior type. Some considerations are made on the times of possible origin and growth of the tumor; the complete absence of the increased pulmonary flow in the presence of a large interatrial communication suggested an early beginning of the tumor that, obstructing the defect, might have hindered the hemodynamic derangement.