Bacterial Homolog Research Articles

Phosphorylated Serine Marks Proteins for Degradation By the Mitochondrial Matrix Protease, Clpxp

Mitochondrial ClpXP is an ATP-dependent protease complex in which the ClpX AAA+ chaperone binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. The serine protease ClpXP is located in the mitochondrial matrix and maintains the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is overexpressed in 45% of AML patients. Genetic or chemical inhibition or hyperactivation of ClpXP kills leukemic cells and stem cells in vitro and in vivo. Yet, the degron sequences or post-translational modifications that are targeted by mitochondrial ClpXP remain to be elucidated. In Bacillus subtilis, the bacterial ClpXP homologue degrades proteins containing phospho-arginine. To determine if phosphorylated amino acids also mark proteins for mitochondrial ClpXP degradation, we evaluated human ClpXP protease activity in the presence of phosphorylated amino acids. Recombinant human ClpXP was incubated with FITC-tagged casein substrates and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr). The release of fluorogenic FITC-tag was measured to assess ClpXP protease activity. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP. Similar inhibition was seen with short peptides containing pSer and pThr. In contrast, pTyr, pArg, free phosphates, and the dephosphorylated amino acids or peptides did not affect ClpXP protease activity in this assay. Although pSer and pThr inhibited the protease activity of ClpXP, they had no impact on ClpX ATPase activity nor the peptidase activity of isolated ClpP. We characterized ClpP peptidase activity and ClpX ATPase activity using the Ac-Trp-Leu-Ala-AMC (Ac-WLA-AMC) peptidase activity assay and the Malachite Green Phosphate assay, respectively. pSer and pThr had no effect on ClpP peptidase activity nor ClpX ATPase activity. Therefore, pSer and pThr specifically inhibited ClpXP's ability to degrade full-length proteins. Next, we investigated if pSer and pThr could bind to ClpX or ClpP. Thermal shift binding assays indicated that pSer and pThr but not pTyr and pArg bound to ClpX and none of the phosphorylated amino acids bound to ClpP. Similarly, Ala-pSer-Ala (ApSA), Arg-Arg-Ala-pSer-Val-Ala (RRApSVA) and Ala-pThr-Ala (ApTA) peptides bound to ClpX, while the non-phosphorylated peptides did not. Thus, taken together, pSer and pThr interact with ClpX at a site outside its ATP binding pocket. Previously we showed that ClpX and ClpP interact with respiratory chain complex II subunit SDHA. Moreover, inhibition of ClpP in AML cells increased misfolded or damaged SDHA protein and impaired respiratory chain complex II activity. Therefore, we asked if inhibition of ClpP and ClpX impacted levels of SDHA serine phosphorylation. We knocked down ClpX and ClpP individually in OCI-AML2 cells using shRNA. Then, mitochondrial lysates were prepared, serine phosphorylated proteins were immunoprecipitated and probed for SDHA. Increased abundance of serine phosphorylated SDHA was observed in AML cells after ClpP and ClpX knockdown. We also demonstrated that addition of recombinant ClpXP to the immunoprecipitates selectively degraded the fraction of serine phosphorylated SDHA. We then showed that serine phosphorylated SDHA was present only in the digitonin insoluble fraction of mitochondria proteins, indicating it is only found on damaged protein aggregates. In summary, we discovered that pSer and pThr promote the degradation of damaged respiratory chain proteins by mitochondrial ClpXP. Thus, this work highlights new mitochondrial biology and identifies a potential new strategy to develop ClpXP inhibitors for the treatment of AML.

Anionic lipids unlock the gates of select ion channels in the pacemaker family.

Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.

Evolution of an Iron-Detoxifying Protein: Eukaryotic and Rickettsia Frataxins Contain a Conserved Site Which Is Not Present in Their Bacterial Homologues.

Friedreich's ataxia is a neurodegenerative disease caused by mutations in the frataxin gene. Frataxin homologues, including bacterial CyaY proteins, can be found in most species and play a fundamental role in mitochondrial iron homeostasis, either promoting iron assembly into metaloproteins or contributing to iron detoxification. While several lines of evidence suggest that eukaryotic frataxins are more effective than bacterial ones in iron detoxification, the residues involved in this gain of function are unknown. In this work, we analyze conservation of amino acid sequence and protein structure among frataxins and CyaY proteins to identify four highly conserved residue clusters and group them into potential functional clusters. Clusters 1, 2, and 4 are present in eukaryotic frataxins and bacterial CyaY proteins. Cluster 3, containing two serines, a tyrosine, and a glutamate, is only present in eukaryotic frataxins and on CyaY proteins from the Rickettsia genus. Residues from cluster 3 are blocking a small cavity of about 40 Å present in E. coli's CyaY. The function of this cluster is unknown, but we hypothesize that its tyrosine may contribute to prevent formation of reactive oxygen species during iron detoxification. This cluster provides an example of gain of function during evolution in a protein involved in iron homeostasis, as our results suggests that Cluster 3 was present in the endosymbiont ancestor of mitochondria and was conserved in eukaryotic frataxins.

Viral origin of eukaryotic type IIA DNA topoisomerases.

Type II DNA topoisomerases of the family A (Topo IIAs) are present in all Bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation, and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologs and have no orthologs in Archaea. Interestingly, eukaryotic Topo IIAs have close homologs in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses. Topo IIAs are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses on a dataset combining viral and cellular Topo IIA homologs. Topo IIAs encoded by Bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both Bacteria and Caudoviricetes from the data set, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long-branch attraction artifact. The topologies of our trees suggest that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.

Isolation and structure of the fibril protein, a major component of the internal ribbon for Spiroplasma swimming.

Spiroplasma, which are known pathogens and commensals of arthropods and plants, are helical-shaped bacteria that lack a peptidoglycan layer. Spiroplasma swim by alternating between left- and right-handed helicity. Of note, this system is not related to flagellar motility, which is widespread in bacteria. A helical ribbon running along the inner side of the helical cell should be responsible for cell helicity and comprises the bacterial actin homolog, MreB, and a protein specific to Spiroplasma, fibril. Here, we isolated the ribbon and its major component, fibril filament, for electron microscopy (EM) analysis. Single-particle analysis of the fibril filaments using the negative-staining EM revealed a three-dimensional chain structure composed of rings with a size of 11 nm wide and 6 nm long, connected by a backbone cylinder with an 8.7 nm interval with a twist along the filament axis. This structure was verified through EM tomography of quick-freeze deep-etch replica sample, with a focus on its handedness. The handedness and pitch of the helix for the isolated ribbon and fibril filament agreed with those of the cell in the resting state. Structures corresponding to the alternative state were not identified. These results suggest that the helical cell structure is supported by fibril filaments; however, the helical switch is caused by the force generated by the MreB proteins. The isolation and structural outline of the fibril filaments provide crucial information for an in-depth clarification of the unique swimming mechanism of Spiroplasma.

Clinical Characteristics and Risk Factors for Intra-Abdominal Infection with Chryseobacterium indologenes after Orthotopic Liver Transplantation.

The incidence of hospital-acquired infections caused by Chryseobacterium indologenes (C. indologenes) is increasing. This study investigated the epidemiological and clinical features of C. indologenes intra-abdominal infections in patients who underwent orthotopic liver transplantation (OLT). In this retrospective study, 53 consecutive non-replicate clinical isolates of C. indologenes were collected and identified from the OLT patients at a tertiary care university hospital in Shanghai in 2017. Genetic relatedness of the isolates was determined by enterobacterial repetitive intergenic consensus polymerase chain reaction DNA fingerprinting. Antimicrobial susceptibility of the isolates was measured using the microdilution broth method. Nosocomial clonal transmission of C. indologenes was confirmed by bacterial homology analysis. All C. indologenes isolates were resistant to β-lactams, carbapenems, quinolones, and aminoglycosides, and showed susceptibility to trimethoprim–sulfamethoxazole and minocycline. Multivariate risk modelling revealed that ≥2 bed transfers and an operation time of ≥8 h were independent risk factors for C. indologenes intra-abdominal infection after OLT. A nomogram was constructed based on the screened risk factors, which showed good concordance and accuracy. Clonal dissemination of C. indologenes in OLT patients was demonstrated and several risk factors for intra-abdominal infections were identified. Epidemiological surveillance of this organism and extensive surveillance programs are imperative worldwide.

Crystal structure and reaction mechanism of a bacterial Mg-dechelatase homolog from the Chloroflexi Anaerolineae.

Chlorophyll degradation plays a myriad of physiological roles in photosynthetic organisms, including acclimation to light environment and nutrient remobilization during senescence. Mg extraction from chlorophyll a is the first and committed step of the chlorophyll degradation pathway. This reaction is catalyzed by the Mg-dechelatase enzyme encoded by Stay-Green (SGR). The reaction mechanism of SGR protein remains elusive since metal ion extraction from organic molecules is not a common enzymatic reaction. Additionally, experimentally derived structural information about SGR or its homologs has not yet been reported. In this study, the crystal structure of the SGR homolog from Anaerolineae bacterium was determined using the molecular replacement method at 1.85 Å resolution. Our previous study showed that three residues-H32, D34, and D62 are essential for the catalytic activity of the enzyme. Biochemical analysis involving mutants of D34 residue further strengthened its importance in the functioning of the dechelatase. Docking simulation also revealed the interaction between the D34 side chain and central Mg ion of chlorophyll a. Structural analysis showed the arrangement of D34/H32/D62 in the form of a catalytic triad that is generally found in hydrolases. The probable reaction mechanism suggests that deprotonated D34 side chain coordinates and destabilizes Mg, resulting in Mg extraction. Besides, H32 possibly acts as a general base catalyst and D62 facilitates H32 to be a better proton acceptor. Taken together, the reaction mechanism of SGR partially mirrors the one observed in hydrolases.

Роль білків теплового шоку в прогнозуванні перебігу клімактеричного синдрому

The problem of menopause has been relevant for many decades. A pathogenetically justified method of treatment, taking into account the change in the hormonal background of a woman, is hormone replacement therapy. Determination of immune markers of cellular homeostasis is one of the promising directions, therefore, the study of changes in the levels of antibodies to HSP60 can become a reliable marker of both the course and compensation of disorders of the neurovegetative state in perimenopause. Purpose - to study the role of anti-Müllerian, follicle-stimulating, luteinizing, thyroid-stimulating hormones, antibodies (IgG) to human heat shock protein 60 (HSP60) and its bacterial homologue (GroEl), and obstetric history on the diagnosis and treatment of vasomotor symptoms in perimenopausal women. Materials and methods. 158 patients with climacteric syndrome in perimenopause were examined in the period October 2019 - March 2022. The Group 1 (main) included 80 patients who agreed to treatment and received it according to the standard scheme: combined treatment in a cyclical regimen - estrogens (17β-estradiol) + progestogens (dydrogesterone); the Group 2 (comparison) included 78 women who refused treatment due to estrogen deficiency. Results. Revealed a decrease (p<0.01) in the risk of not achieving a high treatment effect with vasomotor symptoms for treated patients, OR=0.04 (95% CI: 0.01-0.18) compared to the group of untreated patients. With the number of deliveries, the risk of not achieving a high treatment effect in women with vasomotor symptoms increases (p=0.01), OR=2.27 (95% CI: 1.2-4.27) for each delivery. A lower (p=0.039) risk of not achieving a high treatment effect was found in women with vasomotor symptoms at a higher GroEl level, ОR=0.994 (95% CI: 0.987-1.000) for each ng. When standardizing by the thyrotropin hormone and the manifestation of vasomotor symptoms before or without treatment, a decrease (p<0.001) in the risk of not achieving a high effect of treatment in women with vasomotor symptoms for treated patients was found, OR=0.04 (95% CI: 0.01-0.20). Conclusions. This study found that there is no informative indicator for predicting the effectiveness of treatment in women with vasomotor symptoms. It was established that the HSP60 indicator does not affect, and the GroEl indicator affects the effectiveness of the treatment of vasomotor clinical manifestations. The research was carried out in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the Local Ethics Committee of the institutions. The informed consent of the women was obtained for conducting the studies. No conflict of interest was declared by the authors.

Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons.

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, −4, and −5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, −4, and −5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and −4 or TRPC1 and −5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, −4, and −5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.

Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50

DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3' → 5' exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed.

The SOS response-associated peptidase (SRAP) domain of YedK catalyzes ring opening of abasic sites and reversal of its DNA–protein cross-link

Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA–protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3′-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.

Investigating the effect of bacteriophages on bacterial FtsZ localisation.

Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Several agents that block cell division have been shown to mislocalise FtsZ, including the bacteriophage λ-encoded Kil peptide, resulting in defective cell division and a filamentous phenotype, making FtsZ an attractive target for antimicrobials. In this study, we have used an in vitro meningitis model system for studying the effect of bacteriophages on FtsZ using fluorescent E. coli EV36/FtsZ-mCherry and K12/FtsZ-mNeon strains. We show localisation of FtsZ to the bacterial cell midbody as a single ring during normal growth conditions, and mislocalisation of FtsZ producing filamentous multi-ringed bacterial cells upon addition of the known inhibitor Kil peptide. We also show that when bacteriophages K1F-GFP and T7-mCherry were applied to their respective host strains, these phages can inhibit FtsZ and block bacterial cell division leading to a filamentous multi-ringed phenotype, potentially delaying lysis and increasing progeny number. This occurs in the exponential growth phase, as actively dividing hosts are needed. We present that ZapA protein is needed for phage inhibition by showing a phenotype recovery with a ZapA mutant strain, and we show that FtsI protein is also mislocalised upon phage infection. Finally, we show that the T7 peptide gp0.4 is responsible for the inhibition of FtsZ in K12 strains by observing a phenotype recovery with a T7Δ0.4 mutant.

Profiling a Community-Specific Function Landscape for Bacterial Peptides Through Protein-Level Meta-Assembly and Machine Learning.

Small proteins, encoded by small open reading frames, are only beginning to emerge with the current advancement of omics technology and bioinformatics. There is increasing evidence that small proteins play roles in diverse critical biological functions, such as adjusting cellular metabolism, regulating other protein activities, controlling cell cycles, and affecting disease physiology. In prokaryotes such as bacteria, the small proteins are largely unexplored for their sequence space and functional groups. For most bacterial species from a natural community, the sample cannot be easily isolated or cultured, and the bacterial peptides must be better characterized in a metagenomic manner. The bacterial peptides identified from metagenomic samples can not only enrich the pool of small proteins but can also reveal the community-specific microbe ecology information from a small protein perspective. In this study, metaBP (Bacterial Peptides for metagenomic sample) has been developed as a comprehensive toolkit to explore the small protein universe from metagenomic samples. It takes raw sequencing reads as input, performs protein-level meta-assembly, and computes bacterial peptide homolog groups with sample-specific mutations. The metaBP also integrates general protein annotation tools as well as our small protein-specific machine learning module metaBP-ML to construct a full landscape for bacterial peptides. The metaBP-ML shows advantages for discovering functions of bacterial peptides in a microbial community and increases the yields of annotations by up to five folds. The metaBP toolkit demonstrates its novelty in adopting the protein-level assembly to discover small proteins, integrating protein-clustering tool in a new and flexible environment of RBiotools, and presenting the first-time small protein landscape by metaBP-ML. Taken together, metaBP (and metaBP-ML) can profile functional bacterial peptides from metagenomic samples with potential diverse mutations, in order to depict a unique landscape of small proteins from a microbial community.

Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria.

Vibrio cholerae biotype El Tor is perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and VSP-2 distinguish El Tor from previous pandemic V. cholerae strains. Using a co-occurrence analysis of VSP genes in >200,000 bacterial genomes we built gene networks to infer biological functions encoded in these islands. This revealed that dncV, a component of the cyclic-oligonucleotide-based anti-phage signalling system (CBASS) anti-phage defence system, co-occurs with an uncharacterized gene vc0175 that we rename avcD for anti-viral cytodine deaminase. We show that AvcD is a deoxycytidylate deaminase and that its activity is post-translationally inhibited by a non-coding RNA named AvcI. AvcID and bacterial homologues protect bacterial populations against phage invasion by depleting free deoxycytidine nucleotides during infection, thereby decreasing phage replication. Homologues of avcD exist in all three domains of life, and bacterial AvcID defends against phage infection by combining traits of two eukaryotic innate viral immunity proteins, APOBEC and SAMHD1.

Solid-state NMR 13C and 15 N resonance assignments of Vibrio sp. SemiSWEET transporter in lipid bilayers

The Sugar Will Eventually be Exported Transporter (SWEET) family is a new class of transporters that plays crucial roles in the cellular sugar transport process. Their bacterial homologs, known as SemiSWEETs, are among the smallest transporters and can be used as a prototype for studying the biological properties of sugar transporters. Here, a set of dipolar-based multidimensional solid-state NMR spectra were employed to investigate the structure of Vibrio sp. SemiSWEET (Vs-SemiSWEET) reconstituted in the native-like lipid bilayers. A nearly complete (88% of the amino acid residues) backbone and side-chain 13C and 15N chemical shift assignments of Vs-SemiSWEET were obtained. The overall secondary structure of Vs-SemiSWEET predicted from the obtained 13C and 15N chemical shifts is similar to that from X-ray crystallography, with some differences, reflecting the influence of the membrane environments to the structure of membrane proteins.

The Evolutionary History of Peptidases Involved in the Processing of Organelle-Targeting Peptides.

Most of the proteins present in mitochondria and chloroplasts, the organelles acquired via endosymbiotic events, are encoded in the nucleus and translated into the cytosol. Most of such nuclear-encoded proteins are specifically recognized via an N-terminal-encoded targeting peptide (TP) and imported into the organelles via a translocon machinery. Once imported, the TP is degraded by a succession of cleavage steps ensured by dedicated peptidases. Here, we retrace the evolution of the families of the mitochondrial processing peptidase (MPP), stromal processing peptidase (SPP), presequence protease (PreP), and organellar oligo-peptidase (OOP) that play a central role in TP processing and degradation across the tree of life. Their bacterial distributions are widespread but patchy, revealing unsurprisingly complex history of lateral transfers among bacteria. We provide evidence for the eukaryotic acquisition of MPP, OOP, and PreP by lateral gene transfers from bacteria at the time of the mitochondrial endosymbiosis. We show that the acquisition of SPP and of a second copy of OOP and PreP at the time of the chloroplast endosymbiosis was followed by a differential loss of one PreP paralog in photosynthetic eukaryotes. We identified some contrasting sequence conservations between bacterial and eukaryotic homologs that could reflect differences in the functional context of their peptidase activity. The close vicinity of the eukaryotic peptidases MPP and OOP to those of several bacterial pathogens, showing antimicrobial resistance, supports a scenario where such bacteria were instrumental in the establishment of the proteolytic pathway for TP degradation in organelles. The evidence for their role in the acquisition of PreP is weaker, and none is observed for SPP, although it cannot be excluded by the present study.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.

Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase

Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the −10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the −10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson’s disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.