Long, unique cytoskeleton-like structures resembling microtubules of higher eukaryotes were discovered in Mycoplasmoides gallisepticum (former taxonomy name Mycoplasma gallisepticum), one of the smallest prokaryotes. Identifying the structural components of these formations is of great interest. These structures may contribute to the motility of M. gallisepticum cells. Since proteins involved in motility are potential targets for new antibacterial therapies, studying them is crucial. M. gallisepticum causes chronic respiratory diseases in poultry, leading to significant economic losses. Therefore, combating this mycoplasma holds serious practical importance. Here, using immunoprecipitation with anti-tubulin antibodies and mass spectrometry, a previously undetected "tubulin-like" protein in the M. gallisepticum cell lysate was identified as type I glyceraldehyde-3-phosphate dehydrogenase (GapD). Moreover, this protein was one of the 10 major components of the Triton-insoluble fraction of the M. gallisepticum proteins. Bioinformatics analysis revealed both similarities and differences between the primary and spatial structures of tubulins and GapD. While GapD is not a classical tubulin-like protein, it is the only Triton-insoluble component exhibiting certain tubulin-like characteristics. The pure GapD protein formed loose filament-like structures in KGA buffer. Finally, GapD was directly recognized by TU-01 antibodies in its recombinant and native forms. Therefore, GapD may be considered as a candidate for the role of a protein involved in the formation of cytoskeleton-like structures, with functions extending beyond its traditional role in glycolysis. Similar to certain enzymes found in Mycoplasma mobile, other Triton-insoluble proteins in M. gallisepticum, although primarily enzymatic, are also likely candidates for components of cytoskeleton-like structures.

In the original publication [...].

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ's attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.

Bacteriophage ΦKZ is the founding member of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa , is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems the host already possesses. On infection, it forms a “nucleus”, erecting a barrier around its massive genome to exclude host restriction endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial β/β′ subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to 3.3 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, with structural homology and a biochemical role implying that it has evolved from an ancestral homologue to σ-factor.

Bacillus subtilis bacteriophage AR9 employs two strategies for efficient host takeover control and host antiviral defense evasion – it encodes two unique DNA-dependent RNA polymerases (RNAPs) that function at different stages of virus morphogenesis in the cell, and its double stranded (ds) DNA genome contains uracils instead of thymines throughout1,2. Unlike any known RNAP, the AR9 non-virion RNAP (nvRNAP), which transcribes late phage genes, recognizes promoters in the template strand of dsDNA and efficiently differentiates obligatory uracils from thymines in its promoters3. Here, using structural data obtained by cryo-electron microscopy and X-ray crystallography on the AR9 nvRNAP core, holoenzyme, and a promoter complex, and a variety of in vitro transcription assays, we elucidate a unique mode of uracil-specific, template strand-dependent promoter recognition. It is achieved by a tripartite interaction between the promoter specificity subunit, the core enzyme, and DNA adopting a unique conformation. We also show that interaction with the non-template strand plays a critical role in the process of AR9 nvRNAP promoter recognition in dsDNA, and that the AR9 nvRNAP core and a part of its promoter specificity subunit that interacts with the core are structurally similar to their bacterial RNAP counterparts. Our work demonstrates the extent to which viruses can evolve new functional mechanisms to control acquired multisubunit cellular enzymes and make these enzymes serve their needs.

Protealysin is a thermolysin‐like protease of Serratia proteamaculans capable of specifically cleaving actin, which correlates with the invasive activity of these bacteria. Here, we show that inactivation of the protealysin gene does not inhibit invasion but, in contrast, leads to a twofold increase in the S. proteamaculans invasive activity. By mass spectrometry, we identified the outer membrane protein OmpX as a substrate of protealysin. Recombinant E. coli carrying the OmpX gene truncated by 40 N‐terminal residues or both the OmpX and protealysin genes, in contrast to the full‐length OmpX, do not increase adhesion of these bacteria, indicating that the 40 N‐terminal residues of OmpX are indispensable for S. proteamaculans invasion. Our results show that both protealysin and its substrates can stimulate Serratia invasion.

The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits β2 and β3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of β3. Interestingly, succinylation of β6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the β3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.

The article analyzes the main views on the future of humans and their health. The idea discussed in the paper that the most promising anthropological paradigm is the view of a human as a creature with significant latent evolutionary potential. The authors substantiate the existence of the phenomenon of “reviving innovation.” Up to this date, it is most clearly represented in the field of agriculture and medicine, when the most progressive are traditional knowledge associated with a new technological structure and scientific research. Empirical arguments are made in favor of the position that opposes transhumanism. The author argues that the phenomenon of reviving innovation is widespread in culture. This innovation just allows one to show the hidden evolutionary potential in a person while maintaining his/her identity.

The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 ( Pou5f1 ) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018–1029

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Non-canonical multisubunit DNA-dependent RNA-polymerases (RNAP) form a new group of the main transcription enzymes, which have only distinct homology to the catalytic subunits of canonical RNAPs of bacteria, archaea and eukaryotes. One of the rare non-canonical RNAP, which was partially biochemically characterized, is non-virion RNAP (nvRNAP) encoded by Pseudomonas phage phiKZ. PhiKZ nvRNAP consists of five subunits, four of which are homologs of β and β’ subunit of bacterial RNAP, and the fifth subunits with unknown function. To understand the role of the fifth subunit in phiKZ nvRNAP, we created co-expression system allowing to get recombinant full five-subunit (5s) and four-subunit (4s) complexes and performed their comparison. The 5s recombinant complex is active on phage promoters in vitro as the native nvRNAP. The 4s complex cannot extend RNA, so 4s complex is not a catalytically active core of phiKZ nvRNAP. Thus, the phiKZ fifth subunit is not only a promoter-recognition subunit, but it plays an important role in the formation of active phiKZ nvRNAP.

Линкерный гистон H1 - один из основных ядерных белков, принимающих участие в структурно регуляторной организации хроматина. Ряд данных указывает на то, что посттрансляционные модификации H1 способны модулировать активность хроматина. С помощью масс-спектрометрии высокого разрешения (MALDI-FT-ICR-MS) мы обнаружили существенные различия в природе и положениях посттрансляционных модификаций в некоторых вариантах гистона H1 (H1.3-H1.5) из эмбриональных стволовых клеток и дифференцированных клеток - иммортализованных фибробластов линии NIH/3T3 и эмбриональных фибробластов (МЭФ). Так, метилирование K75 в вариантах H1.2-1.4, метилирование K108, K148, K151, K152 K154, K155, K160, K161, K179 и K185 в Н1.1, а также K168 в H1.2, фосфорилирование S129, T146, T149, S159, S163 и S180 в H1.1, T180 в H1.2 и T155 в H1.3 идентифицированы исключительно в эмбриональных стволовых клетках. Большинство сайтов ацетилирования вариантов H1.0 и H1.2 в эмбриональных стволовых клетках расположены в C-концевых доменах, известных своим участием в стабилизации конденсированного хроматина. Полученные данные могут послужить основой для дальнейших исследований, направленных на анализ функциональной значимости посттрансляционной модификации гистона H1 для процессов самообновления и дифференцировки плюрипотентных стволовых клеток.

The photo- and cathodoluminescence spectra (PL, CL) of diamond single crystals synthesized under high-pressure and at high-temperature (HPHT sintering) from detonation nanodiamonds (DND) particles with Gd and Eu ion-functionalized surfaces have been studied. The HPHT sintering was made under pressures (P congruent to 7 GPa) and temperature (T = 1300 - 1500 degrees C). The DND particles have sizes of 4 - 5 nm, hydrocarbons and/or alcohols were used at the HPHT synthesis instead of traditional metal catalysts. The initial and synthesized crystals were characterized by X-ray microanalysis, mass-spectroscopy, scanning electron microscopy (SEM), PL and CL. Mass spectra confirmed that Gd and Eu atoms were inserted into the volume of diamond single crystals in the HPHT sintering. A significant conversion of the PL and CL spectra of synthesized diamond single crystals compared with HPHT diamond crystals were observed. Transformation of the point defect assemblies inherent to the diamond crystal can explain the observed spectra.

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.