Synthesis Of Subunits Research Articles

Total Synthesis of Marformycins A and D.

We report the synthesis of the antimicrobial cyclodepsipeptides marformycin A (1) and marformycin D (2) using a solid-phase approach. A scalable solution-phase synthesis of the γ-hydroxypiperazic acid subunit in 2, starting from cis-hydroxyproline, is also described. Structural analysis of 1 and its Leu-epi congener demonstrates conformational differences that may underlie their divergent antimicrobial activities. The described approach enables further development of conformation-activity relationships within this class of depsipeptide natural products.

Multi-Lasso Peptide-Based Synergistic Nanocomposite: A High-Stability, Broad-Spectrum Antimicrobial Agent with Potential for Combined Antibacterial Therapy.

Lasso peptides, natural biological microcins composed of small molecules, have demonstrated efficient bactericidal activity. However, a single lasso peptide is characterized by a narrow and targeted bactericidal spectrum. In this study, a chitosan (CN) derivative-based polymer nanomaterial incorporating three lasso peptides (MccY, MccJ25, and Klebsidin) was designed and synthesized to broaden its antimicrobial spectrum. To enhance resistance to acid and alkali conditions, arginine was appended to the terminus of conjugates, resulting in Chitosan-Lasso-Peptides-Arg (CN-LPs-Arg), and the nanomaterial biocompatibility and bactericidal activity were characterized. Chemical stability test results demonstrate that CN-LPs-Arg effectively buffered the acid-base effect of the compound. Notably, CN-LPs-Arg extended the antimicrobial spectrum of Gram-negative and Gram-positive strains including Klebsiella, Salmonella, and Staphylococcus (MIC = 0.01-1.0 μM). CN-LPs-Arg exerts its destructive effects on bacteria via a series of mechanisms; it adheres to and then penetrates the membrane, causes rupture, and leads to bacterial death. Transcriptomic data revealed that CN-LPs-Arg produced a distinct inhibitory effect on ribosomal protein subunits synthesis pathways and membrane metabolic inhibition. Furthermore, CN-LPs-Arg was nontoxic to cells and exhibited excellent biocompatibility. CN-LPs-Arg reduced bacterial burden in organs and the levels of inflammatory factors IL-6, IL-8, and TNF-α in tissues of mice with acute bacterial infections. Furthermore, it promoted the recovery of Klebsiella-infected C57BL/6 mice, demonstrating a favorable therapeutic effect in vivo. The multilasso peptide-based synergistic nanocomposite of CN-LPs-Arg exhibited high stability as a broad-spectrum antimicrobial agent with potential for combined antibacterial therapy and utilization in the fields of food, biomedicine, and public health.

A molecular genetic toolkit for the differential expression analysis of soybean β-conglycinin subunit genes

The majority of proteins in soybean seeds are storage ones, including β-conglycinin and glycinin, which are necessary for seed germination. At the same time, they are the most valuable soy proteins used in the food industry, since their subunit composition and proportion of total protein can affect the quality of the resulting food product. β-conglycinins are trimers with different composition of subunits which are designated as α', α, β and encoded by the CG-1, CG-3, and CG-4 genes, respectively. The PCR analysis employed a model soybean cultivar ‘Sentyabrinka’. A complementary DNA synthesized from the RNA isolated from seeds of the studied cultivar served as a template. The in silico created pairs of primers for CG-1, CG-3, and CG-4 gene transcripts were used. As the result of PCR and the analysis of the obtained electrophoregrams, optimal annealing temperatures of primers for the CG-1, CG-3 and CG-4 genes were selected, at which only the characteristic fragment was observed. Thus, a molecular genetic toolkit has been developed for a comprehensive study of the qualitative and quantitative composition of soybean protein and can be used for further analysis of differential expression of genes responsible for the synthesis of β-conglycinin subunits.

Mechanisms of mitochondrial resilience in teleostean radial glia under hypoxic stress

Radial glial cells (RGCs) are remarkable cells, essential for normal development of the vertebrate central nervous system. In teleost fishes, RGCs play a pivotal role in neurogenesis and regeneration of injured neurons and glia. RGCs also exhibit resilience to environmental stressors like hypoxia via metabolic adaptations. In this study, we assessed the physiology of RGCs following varying degrees of hypoxia, with an emphasis on reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), mitophagy, and energy metabolism. Our findings demonstrated that hypoxia significantly elevated ROS production and induced MMP depolarization in RGCs. The mitochondrial disturbances were closely associated with increased mitophagy, based on the co-localization of mitochondria and lysosomes. Key mitophagy-related genes were also up-regulated, including those of the BNIP3/NIX mediated pathway as well as the FUNDC1 mediated pathway. Such responses suggest robust cellular mechanisms are initiated to counteract mitochondrial damage due to increasing hypoxia. A significant metabolic shift from oxidative phosphorylation to glycolysis was also observed in RGCs, which may underlie an adaptive response to sustain cellular function and viability following a reduction in oxygen availability. Furthermore, hypoxia inhibited the synthesis of mitochondrial complexes subunits in RGCs, potentially related to elevated HIF-2α expression with 3 % O2. Taken together, RGCs appear to exhibit complex adaptive responses to hypoxic stress, characterized by metabolic reprogramming and the activation of mitophagy pathways to mitigate mitochondrial dysfunction.

Importance of conserved hydrophobic pocket region in yeast mitoribosomal mL44 protein for mitotranslation and transcript preference

The mitochondrial ribosome (mitoribosome) is responsible for the synthesis of key oxidative phosphorylation subunits encoded by the mitochondrial genome. Defects in mitoribosomal function therefore can have serious consequences for the bioenergetic capacity of the cell. Mutation of the conserved mitoribosomal mL44 protein has been directly linked to childhood cardiomyopathy and progressive neurophysiology issues. To further explore the functional significance of the mL44 protein in supporting mitochondrial protein synthesis we have performed a mutagenesis study of the yeast mL44 homolog, the MrpL3/mL44 protein. We specifically investigated the conserved hydrophobic pocket region of the MrpL3/mL44 protein, where the known disease-related residue in the human mL44 protein (L156R) is located. While our findings identify a number of residues in this region critical for MrpL3/mL44’s ability to support the assembly of translationally active mitoribosomes, the introduction of the disease-related mutation into the equivalent position in the yeast protein (residue A186) was found not have a major impact on function. The human and yeast mL44 proteins share many similarities in sequence and structure, however results presented here indicate that these two proteins have diverged somewhat in evolution. Finally, we observed that mutation of the MrpL3/mL44 does not impact the translation of all mitochondrial encoded proteins equally, suggesting the mitochondrial translation system may exhibit a transcript hierarchy and prioritization.

The catalytic activity of methyltransferase METTL15 is dispensable for its role in mitochondrial ribosome biogenesis

ABSTRACT Ribosomes are large macromolecular complexes composed of both proteins and RNA, that require a plethora of factors and post-transcriptional modifications for their biogenesis. In human mitochondria, the ribosomal RNA is post-transcriptionally modified at ten sites. The N4-methylcytidine (m4C) methyltransferase, METTL15, modifies the 12S rRNA of the small subunit at position C1486. The enzyme is essential for mitochondrial protein synthesis and assembly of the mitoribosome small subunit, as shown here and by previous studies. Here, we demonstrate that the m4C modification is not required for small subunit biogenesis, indicating that the chaperone-like activity of the METTL15 protein itself is an essential component for mitoribosome biogenesis.

Structure, Regulation, and Significance of Cyanobacterial and Chloroplast Adenosine Triphosphate Synthase in the Adaptability of Oxygenic Photosynthetic Organisms.

In cyanobacteria and chloroplasts (in algae and plants), ATP synthase plays a pivotal role as a photosynthetic membrane complex responsible for producing ATP from adenosine diphosphate and inorganic phosphate, utilizing a proton motive force gradient induced by photosynthesis. These two ATP synthases exhibit similarities in gene organization, amino acid sequences of subunits, structure, and functional mechanisms, suggesting that cyanobacterial ATP synthase is probably the evolutionary precursor to chloroplast ATP synthase. In this review, we explore the precise synthesis and assembly of ATP synthase subunits to address the uneven stoichiometry within the complex during transcription, translation, and assembly processes. We also compare the regulatory strategies governing ATP synthase activity to meet varying energy demands in cyanobacteria and chloroplasts amid fluctuating natural environments. Furthermore, we delve into the role of ATP synthase in stress tolerance and photosynthetic carbon fixation efficiency in oxygenic photosynthetic organisms (OPsOs), along with the current researches on modifying ATP synthase to enhance carbon fixation efficiency under stress conditions. This review aims to offer theoretical insights and serve as a reference for understanding the functional mechanisms of ATP synthase, sparking innovative ideas for enhancing photosynthetic carbon fixation efficiency by utilizing ATP synthase as an effective module in OPsOs.

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

SnapShot: Eukaryotic ribosome biogenesis II

Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis. To view this SnapShot, open or download the PDF.

Acute accumulation of PIM2 and NRF2 and recovery of β5 subunit activity mitigate multiple myeloma cell susceptibility to proteasome inhibitors.

Resistance to proteasome inhibitors (PIs) has emerged as an important clinical issue. We investigated the mechanisms underlying multiple myeloma (MM) cell resistance to PIs. To mimic their pharmacokinetic/pharmacodynamic (PK/PD) profiles, MM cells were treated with bortezomib and carfilzomib for 1h at concentrations up to 400 and 1,000nM, respectively. Susceptibility to these PIs markedly varied among MM cell lines. Pulsatile treatments with PIs suppressed translation, as demonstrated by incorporation of puromycin at 24h in PI-susceptible MM.1S cells, but not PI-resistant KMS-11 cells. Inhibition of β5 subunit activity decreased at 24h in KMS-11 cells, even with the irreversible PI carfilzomib, but not under suppression of protein synthesis with cycloheximide. Furthermore, the proteasome-degradable pro-survival factors PIM2 and NRF2 acutely accumulated in MM cells subjected to pulsatile PI treatments. Accumulated NRF2 was trans-localized into the nucleus to induce the expression of its target gene, HMOX1, in MM cells. PIM and Akt inhibition restored the anti-MM effects of PIs, even against PI-resistant KMS-11 cells. Collectively, these results suggest that increased synthesis of β5 proteasome subunit and acute accumulation of PIM2 and NRF2 reduce the anti-MM effects of PIs.

BRIX1 promotes ribosome synthesis and enhances glycolysis by selected translation of GLUT1 in colorectal cancer.

Ribosome biogenesis protein BRX1 homolog (BRIX1) is critically required for the synthesis of the 60S ribosome subunit. However, the role and mechanism of BRIX1 in colorectal cancer (CRC) remain unclear. Kyoto Encyclopedia of Gene and Genome pathway and Gene Ontology analyses were used for bioinformatics analysis. The rRNA levels were detected in CRC tissues and cells. Nascent RNA synthesis was detected via cellular immunofluorescence. The correlation was analyzed between patient Positron Emission Tomography-Computed Tomography (PET-CT) values and their BRIX1 expression. The extracellular acidification rate (ECAR) and oxygen consumption rate were determined via live metabolic analyses. Polysome fractions were collected for BRIX1 mRNA used in translation. The orthotopic model and Cell Counting Kit-8 (CCK8) assay were used to assess BRIX1 function in CRC. BRIX1 is a core protein involved in ribosome-related pathway changes in CRC. Gene Ontology analysis showed that BRIX1 was primarily enriched in ribosome assembly and ribosome biogenesis pathways. In fresh CRC tissue, rRNA levels (5S, 5.8S, 18S and 28S) were higher in the BRIX1 high-expression group than in the BRIX1 low-expression group. Similarly, BRIX1 knockdown significantly decreased rRNA levels for 5S, 5.8S, 18S and 28S in CRC cells, whereas overexpression of BRIX1 significantly increased these levels. In addition, BRIX1 knockdown inhibited nascent RNA synthesis in CRC cells. In clinical data analysis, BRIX1 expression was related to the glucose uptake in PET-CT. BRIX1 knockdown significantly decreased the ECAR value, glucose uptake and lactic acid production in CRC cells, whereas BRIX1 overexpression significantly increased these. Furthermore, BRIX1 knockdown significantly decreased the protein expression of GLUT1, whereas BRIX1 overexpression significantly increased this; however, expression of BRIX1 mRNA was unaffected in either case. Blocking glycolysis by si-GLUT1 or galactose reversed BRIX1 promotion of glycolysis and cell proliferation in CRC cells.

Synthesis and Evaluation of Compound Targeting α7 and β2 Subunits in Nicotinic Acetylcholinergic Receptor.

Nicotinic acetylcholine receptors (nAChRs) are involved in various central nervous system functions and have also been implicated in several neurodegenerative disorders. The heteromeric α4β2* and homomeric α7 are two major nAChR subtypes which have been studied in the brain using positron emission tomography (PET). Our comparative autoradiographic studies of the two receptor types in the mouse and rat brains show major differences in the thalamus (α4β2* >> α7), hippocampus (α7 >> α4β2*), and subiculum (α4β2* >> α7). A relatively newer heteromeric α7β2 nAChR subtype has been identified in the brain which may have a greater role in neurodegeneration. We report the development of KS7 (3-(2-(S)-azetidinylmethoxy)-5-(1,4-diaza-bicyclo[3.2.2]nonane)pyridine) which incorporates structural features of Nifzetidine (high affinity for α4β2* nAChR) and ASEM (high affinity for α7 nAChR) in an effort to target α7 and β2 subunits in α7β2 nAChR. KS7 exhibited higher affinities (IC50 = 50 to 172 nM) for [3H]cytisine radiolabeled sites and weaker affinities (IC50 = 10 μM) for [125I]-α-bungarotoxin radiolabeled rat brain sites in several brain regions. The weaker affinity of KS7 to α7 nAChR may suggest lack of binding at the α7 subunit of α7β2 nAChR. A radiolabeled derivative of KS7 may be required to identify any specific binding to brain regions suggested to contain α7β2 nAChR.

UGDH promotes tumor-initiating cells and a fibroinflammatory tumor microenvironment in ovarian cancer

BackgroundEpithelial ovarian cancer (EOC) is a global health burden, with the poorest five-year survival rate of the gynecological malignancies due to diagnosis at advanced stage and high recurrence rate. Recurrence in EOC is driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that are supported by a complex extracellular matrix and immunosuppressive microenvironment. To target TICs to prevent recurrence, we identified genes critical for TIC viability from a whole genome siRNA screen. A top hit was the cancer-associated, proteoglycan subunit synthesis enzyme UDP-glucose dehydrogenase (UGDH).MethodsImmunohistochemistry was used to characterize UGDH expression in histological and molecular subtypes of EOC. EOC cell lines were subtyped according to the molecular subtypes and the functional effects of modulating UGDH expression in vitro and in vivo in C1/Mesenchymal and C4/Differentiated subtype cell lines was examined.ResultsHigh UGDH expression was observed in high-grade serous ovarian cancers and a distinctive survival prognostic for UGDH expression was revealed when serous cancers were stratified by molecular subtype. High UGDH was associated with a poor prognosis in the C1/Mesenchymal subtype and low UGDH was associated with poor prognosis in the C4/Differentiated subtype. Knockdown of UGDH in the C1/mesenchymal molecular subtype reduced spheroid formation and viability and reduced the CD133 + /ALDH high TIC population. Conversely, overexpression of UGDH in the C4/Differentiated subtype reduced the TIC population. In co-culture models, UGDH expression in spheroids affected the gene expression of mesothelial cells causing changes to matrix remodeling proteins, and fibroblast collagen production. Inflammatory cytokine expression of spheroids was altered by UGDH expression. The effect of UGDH knockdown or overexpression in the C1/ Mesenchymal and C4/Differentiated subtypes respectively was tested on mouse intrabursal xenografts and showed dynamic changes to the tumor stroma. Knockdown of UGDH improved survival and reduced tumor burden in C1/Mesenchymal compared to controls.ConclusionsThese data show that modulation of UGDH expression in ovarian cancer reveals distinct roles for UGDH in the C1/Mesenchymal and C4/Differentiated molecular subtypes of EOC, influencing the tumor microenvironmental composition. UGDH is a strong potential therapeutic target in TICs, for the treatment of EOC, particularly in patients with the mesenchymal molecular subtype.

Characterising the structural and cellular role of immunoglobulin C-terminal lysine in secretory pathways.

Improved understanding of expression of recombinant immunoglobulin (IgG)-based therapies can decrease manufacturing process costs and bring down costs to patients. Deletion of C-terminal Lysine (C-Lys) from IgG molecules has been shown to greatly impact yield. This study set out to characterise structural components of IgG C-terminal variants which modulate protein expression by examination of the consequences of mutations at the C-terminal of IgG on expression and by the use of fluorescent C-terminal fragment fusion proteins. Cell-based and cell-free experiments were also implemented to characterise how the C-terminal differentially engages with cellular pathways to modulate expression. IgG variants engineered by removal of the C-terminal Lys were expressed at significantly lower rates than control variants by CHO (and HEK) cells. Engineered constructs of mCherry fused with short regions of the C-terminal regions of IgG mimicked the ordering of expressability observed for IgG variants. These fluorescent C-terminal fragment fusions offered the potential to profile how sequences (and point mutations) modified expression. Via combinations of cell and cell-free systems, screening across a range of variants of IgG and mCherry reporter constructs has shown that interactions between specific C-terminal amino acid sequences and the ribosome can regulate the rate and extent of expression. This study highlights the importance of amino acid sequence regulatory events determining the efficiency of production of desirable recombinant proteins, showing that wildtype C-terminal lysine is a necessary capping molecule for IgG1 expression. From a wider perspective, these data are especially significant towards the design of novel entities. The approach has also provided information about novel short C-terminal tags which may be used to provide selective synthesis of specific subunits in the production of multisubunit products. Alternative strategies for removing C-terminal amino acid heterogeneity whilst maintaining efficient rates of expression have been provided.

Changes in the Activity and Content of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes differing, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer’s disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (non-constitutive proteasomes), was shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and protein content in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). In samples from 5xFAD mice, at the age of 380 days, compared with samples from mice of 60 days of age, 4 and 6 times more gene transcripts of the immune subunits PSMB9 and PSMB8 were detected, as well as a significant increase in the number of immune β-subunits (2.8 times – β1i, 2.2 times – β2i) was observed. The results obtained indicate activation of the synthesis of immune subunits and assembly of non-constitutive proteasomes at the terminal stage of pathology development. At the same time, the results of electrophoresis in native conditions indicate the activation of both 20S and 26S proteasomes containing immune subunits in samples from 5xFAD mice, 380 days of age. The obtained data, in combination with available literature, indicate that the activation of non-constitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, that may reflect both the development of neuroinflammation and adaptive processes in tissues.

A traditional gynecological medicine inhibits ovarian cancer progression and eliminates cancer stem cells via the LRPPRC–OXPHOS axis

BackgroundOvarian cancer (OC) is the most lethal malignant gynecological tumor type for which limited therapeutic targets and drugs are available. Enhanced mitochondrial oxidative phosphorylation (OXPHOS), which enables cell growth, migration, and cancer stem cell maintenance, is a critical driver of disease progression and a potential intervention target of OC. However, the current OXPHOS intervention strategy mainly suppresses the activity of the electron transport chain directly and cannot effectively distinguish normal tissues from cancer tissues, resulting in serious side effects and limited efficacy.MethodsWe screened natural product libraries to investigate potential anti-OC drugs that target OXPHOS. Additionally, LC-MS, qRT-PCR, western-blot, clonogenic assay, Immunohistochemistry, wound scratch assay, and xenograft model was applied to evaluate the anti-tumor mechanism of small molecules obtained by screening in OC.ResultsGossypol acetic acid (GAA), a widely used gynecological medicine, was screened out from the drug library with the function of suppressing OXPHOS and OC progression by targeting the leucine-rich pentatricopeptide repeat containing (LRPPRC) protein. Mechanically, LRPPRC promotes the synthesis of OXPHOS subunits by binding to RNAs encoded by mitochondrial DNA. GAA binds to LRPPRC directly and induces LRPPRC rapid degradation in a ubiquitin-independent manner. LRPPRC was overexpressed in OC, which is highly correlated with the poor outcomes of OC and could promote the malignant phenotype of OC cells in vitro and in vivo. GAA management inhibits cell growth, clonal formation, and cancer stem cell maintenance in vitro, and suppresses subcutaneous graft tumor growth in vivo.ConclusionsOur study identified a therapeutic target and provided a corresponding inhibitor for OXPHOS-based OC therapy. GAA inhibits OC progression by suppressing OXPHOS complex synthesis via targeting LRPPRC protein, supporting its potential utility as a natural therapeutic agent for ovarian cancer.

PET117 assembly factor stabilizes translation activator TACO1 thereby upregulates mitochondria-encoded cytochrome C oxidase 1 synthesis

Cytochrome c oxidase, also known as complex IV, facilitates the transfer of electrons from cytochrome c to molecular oxygen, resulting in the production of ATP. The assembly of complex IV is a tightly regulated and intricate process that entails the coordinated synthesis and integration of subunits encoded by the mitochondria and nucleus into a functional complex. Accurate regulation of translation is crucial for maintaining proper mitochondrial function, and defects in this process can lead to a wide range of mitochondrial disorders and diseases. However, the mechanisms governing mRNA translation by mitoribosomes in mammals remain largely unknown. In this study, we elucidate the critical role of PET117, a chaperone protein involved in complex IV assembly, in the regulation of mitochondria-encoded cytochrome c oxidase 1 (COX1) protein synthesis in human cells. Depletion of PET117 reduced mitochondrial oxygen consumption rate and impaired mitochondrial function. PET117 was found to interact with and stabilize translational activator of COX1 (TACO1) and prevent its ubiquitination. TACO1 overexpression rescued the inhibitory effects on mitochondria caused by PET117 deficiency. These findings provide evidence for a novel PET117-TACO1 axis in the regulation of mitochondrial protein expression, and revealed a previously unknown role of PET117 in human cells.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction.

The Terminal Extensions of Dbp7 Influence Growth and 60S Ribosomal Subunit Biogenesis in Saccharomyces cerevisiae

Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis.

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.