Target-locked: A mechanism for disaggregase binding to aggregated proteins.

ClpG is a novel autonomous disaggregase found in Pseudomonas aeruginosa that confers resistance to lethal heat stress. The mechanism by which ClpG specifically targets protein aggregates for disaggregation is unknown. In their recent work published in JBC, Katikaridis et al. (2023) identify an avidity-based mechanism by which four or more ClpG subunits, through specific N-terminal hydrophobic residues located on an exposed ß-sheet loop, interact with multiple hydrophobic patches on an aggregated protein substrate. This study establishes a model for substrate binding to a prokaryotic disaggregase that should inform further investigations into other autonomous disaggregases.

The RavA-ViaA chaperone complex modulates bacterial persistence through its association with the fumarate reductase enzyme.

Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.

On a path toward a broad-spectrum anti-viral: inhibition of HIV-1 and coronavirus replication by SR kinase inhibitor harmine.

IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.

Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules.

ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a self-compartmentalizing protease composed of two stacked heptameric rings that allow protein degradation to occur within the catalytic chamber. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multilayered regulation of ClpP that may present novel ideas for structure-based drug design.

The PAQosome, an R2TP-Based Chaperone for Quaternary Structure Formation.

The Rvb1-Rvb2-Tah1-Pih1/prefoldin-like (R2TP/PFDL) complex is a unique chaperone that provides a platform for the assembly and maturation of many key multiprotein complexes in mammalian cells. Here, we propose to rename R2TP/PFDL as PAQosome (particle for arrangement of quaternary structure) to more accurately represent its unique function.

Analysis of the Evolution of the MoxR ATPases.

MoxR proteins comprise a family of ATPases Associated with diverse cellular Activities (AAA+). These proteins are widespread and found across the diversity of prokaryotic species. Despite their ubiquity, members of the group remain poorly characterized. Only a few examples of MoxR proteins have been associated with cellular roles, where they have been shown to perform chaperone-like functions. A characteristic feature of MoxR proteins is their association with proteins containing the von Willebrand factor type A (VWA) domain. In an effort to understand the spread and diversity of the MoxR family, an evolutionary approach was undertaken. Phylogenetic techniques were used to define nine major subfamilies within the MoxR family. A combination of phylogenetic and genomic approaches was utilized to explore the extent of the partnership between the MoxR and VWA domain containing proteins (VWA proteins). These analyses led to the clarification of genetic linkages between MoxR and VWA proteins. A significant partnership is described here, as seven of nine MoxR subfamilies were found to be linked to VWA proteins. Available genomic data were also used to assess the intraprotein diversification of MoxR and VWA protein sequences. Data clearly indicated that, in MoxR proteins, the ATPase domain is maintained with high conservation while the remaining protein sequence evolves at a faster rate; a similar pattern was observed for the VWA domain in VWA proteins. Overall, our data present insights into the modular evolution of MoxR ATPases.

Leishmania major RUVBL1 has a hexameric conformation in solution and, in the presence of RUVBL2, forms a heterodimer with ATPase activity.

ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.

Reversible inhibition of the ClpP protease via an N-terminal conformational switch.

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from Staphylococcus aureus (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly ß-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of Escherichia coli and Neisseria meningitidis ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.

Multiple functionalities of molecular chaperones revealed through systematic mapping of their interaction networks.

Chaperones are a highly interactive group of proteins that function globally in many cellular processes involved in maintaining protein homeostasis. Traditional biochemical assays typically do not provide a complete view of the intricate networks through which chaperones collaborate to promote proteostasis. Recent advances in high-throughput systematic analyses of chaperone interactions have uncovered that chaperones display a remarkable cooperativity in their interactions with numerous client proteins. This cooperativity has been found to be a fundamental aspect of a properly functioning cell. Aberrant formation or improper regulation of these interactions can easily lead to disease states. Herein, we provide an overview of the use of large-scale interaction assays, whether physical (protein-protein) or genetic (epistatic), to study chaperone interaction networks. Importantly, we discuss the ongoing need for such studies to determine the mechanisms by which protein homeostasis is controlled in the cell.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional.

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.

Recent Advances in Targeting Human Mitochondrial AAA+ Proteases to Develop Novel Cancer Therapeutics.

The mitochondrion is a vital organelle that performs diverse cellular functions. In this regard, the cell has evolved various mechanisms dedicated to the maintenance of the mitochondrial proteome. Among them, AAA+ ATPase-associated proteases (AAA+ proteases) such as the Lon protease (LonP1), ClpXP complex, and the membrane-bound i-AAA, m-AAA and paraplegin facilitate the clearance of misfolded mitochondrial proteins to prevent the accumulation of cytotoxic protein aggregates. Furthermore, these proteases have additional regulatory functions in multiple biological processes that include amino acid metabolism, mitochondria DNA transcription, metabolite and cofactor biosynthesis, maturation and turnover of specific respiratory and metabolic proteins, and modulation of apoptosis, among others. In cancer cells, the increase in intracellular ROS levels promotes tumorigenic phenotypes and increases the frequency of protein oxidation and misfolding, which is compensated by the increased expression of specific AAA+ proteases as part of the adaptation mechanism. The targeting of AAA+ proteases has led to the discovery and development of novel anti-cancer compounds. Here, we provide an overview of the molecular characteristics and functions of the major mitochondrial AAA+ proteases and summarize recent research efforts in the development of compounds that target these proteases.

Mechanisms of acid resistance in Escherichia coli.

Adaptation to acid stress is an important factor in the transmission of intestinal microbes. The enterobacterium Escherichia coli uses a range of physiological, metabolic, and proton-consuming acid resistance mechanisms in order to survive acid stresses as low as pH 2.0. The physiological adaptations include membrane modifications and outer membrane porins to reduce proton influx and periplasmic and cytoplasmic chaperones to manage the effects of acid damage. The metabolic acid resistance systems couple proton efflux to energy generation via select components of the electron transport chain, including cytochrome bo oxidase, NADH dehydrogenase I, NADH dehydrogenase II, and succinate dehydrogenase. Under anaerobic conditions the formate hydrogen lyase complex catalyzes conversion of cytoplasmic protons to hydrogen gas. Finally, each major proton-consuming acid resistance system has a pyridoxal-5'-phosphate-dependent amino acid decarboxylase that catalyzes proton-dependent decarboxylation of a substrate amino acid to product and CO2, and an inner membrane antiporter that exchanges external substrate for internal product.

The Multiple Functions of the PAQosome: An R2TP- and URI1 Prefoldin-Based Chaperone Complex.

The PAQosome (Particle for Arrangement of Quaternary structure) is a large multisubunit chaperone complex that is essential for the assembly and stabilization of other macromolecular complexes. It also interacts with several chaperones including Hsp90, Hsp70, and CCT. The PAQosome is comprised of the R2TP complex, the URI1 prefoldin complex (also known as the non-canonical prefoldin-like complex), the RNA polymerase subunit RPB5, and the WD40 repeat protein WDR92. The R2TP complex is conserved among eukaryotes and has been comprehensively studied over the last 13 years. The R2TP complex is known for its involvement in the assembly and stabilization of L7Ae ribonucleoproteins, U5 small nuclear ribonucleoprotein, RNA polymerase II, phosphatidylinositol-3-kinase-related proteins (PIKKs), and the tuberous sclerosis complex (TSC1-TSC2). By contrast, the URI1 prefoldin complex has evolved exclusively in higher metazoans. Although the URI1 prefoldin complex was initially reported more than 15 years ago, little is known about its function and its role within the PAQosome. Given that URI1 is overexpressed in many types of cancer, it is surprising that the URI1 prefoldin complex has been overlooked. This chapter provides an update on the recent progress uncovering the physiological roles of each PAQosome subunit and provides an overview of the potential functions of the URI1 prefoldin complex.

Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

Dynamics of the ClpP serine protease: a model for self-compartmentalized proteases.

ClpP is a highly conserved serine protease present in most bacterial species and in the mitochondria of mammalian cells. It forms a cylindrical tetradecameric complex arranged into two stacked heptamers. The two heptameric rings of ClpP enclose a roughly spherical proteolytic chamber of about 51 Å in diameter with 14 Ser-His-Asp proteolytic active sites. ClpP typically forms complexes with unfoldase chaperones of the AAA+ superfamily. Chaperones dock on one or both ends of the ClpP double ring cylindrical structure. Dynamics in the ClpP structure is critical for its function. Polypeptides targeted for degradation by ClpP are initially recognized by the AAA+ chaperones. Polypeptides are unfolded by the chaperones and then translocated through the ClpP axial pores, present on both ends of the ClpP cylinder, into the ClpP catalytic chamber. The axial pores of ClpP are gated by dynamic axial loops that restrict or allow substrate entry. As a processive protease, ClpP degrades substrates to generate peptides of about 7-8 residues. Based on structural, biochemical and theoretical studies, the exit of these polypeptides from the proteolytic chamber is proposed to be mediated by the dynamics of the ClpP oligomer. The ClpP cylinder has been found to exist in at least three conformations, extended, compact and compressed, that seem to represent different states of ClpP during its proteolytic functional cycle. In this review, we discuss the link between ClpP dynamics and its activity. We propose that such dynamics also exist in other cylindrical proteases such as HslV and the proteasome.

Novel function discovery with GeneMANIA: a new integrated resource for gene function prediction in Escherichia coli.

MOTIVATION: The model bacterium Escherichia coli is among the best studied prokaryotes, yet nearly half of its proteins are still of unknown biological function. This is despite a wealth of available large-scale physical and genetic interaction data. To address this, we extended the GeneMANIA function prediction web application developed for model eukaryotes to support E.coli. RESULTS: We integrated 48 distinct E.coli functional interaction datasets and used the GeneMANIA algorithm to produce thousands of novel functional predictions and prioritize genes for further functional assays. Our analysis achieved cross-validation performance comparable to that reported for eukaryotic model organisms, and revealed new functions for previously uncharacterized genes in specific bioprocesses, including components required for cell adhesion, iron-sulphur complex assembly and ribosome biogenesis. The GeneMANIA approach for network-based function prediction provides an innovative new tool for probing mechanisms underlying bacterial bioprocesses. CONTACT: gary.bader@utoronto.ca; mohan.babu@uregina.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses.

Substrate Interaction Networks of the Escherichia coli Chaperones: Trigger Factor, DnaK and GroEL.

In the dense cellular environment, protein misfolding and inter-molecular protein aggregation compete with protein folding. Chaperones associate with proteins to prevent misfolding and to assist in folding to the native state. In Escherichia coli, the chaperones trigger factor, DnaK/DnaJ/GrpE, and GroEL/ES are the major chaperones responsible for insuring proper de novo protein folding. With multitudes of proteins produced by the bacterium, the chaperones have to be selective for their substrates. Yet, chaperone selectivity cannot be too specific. Recent biochemical and high-throughput studies have provided important insights highlighting the strategies used by chaperones in maintaining proteostasis in the cell. Here, we discuss the substrate networks and cooperation among these protein folding chaperones.

Structural insights into the inactive subunit of the apicoplast-localized caseinolytic protease complex of Plasmodium falciparum.

The ATP-dependent caseinolytic protease, ClpP, is highly conserved in bacteria and in the organelles of different organisms. In cyanobacteria, plant plastids, and the apicoplast of the genus Plasmodium, a noncatalytic paralog of ClpP, termed ClpR, has been identified. ClpRs are found to form heterocomplexes with ClpP resulting in a ClpRP tetradecameric cylinder having less than 14 catalytic triads. The exact role of ClpR in such a complex remains enigmatic. Here we describe the x-ray crystal structure of ClpR protein heptamer from Plasmodium falciparum (PfClpR). This is the first structure of a ClpR protein. The structure shows that the PfClpR monomer adopts a fold similar to that of ClpP, but has a unique motif, which we named the R-motif, forming a ß turn located near the inactive catalytic triad in a three-dimensional space. The PfClpR heptamer exhibits a more open and flat ring than a ClpP heptamer. PfClpR was localized in the P. falciparum apicoplast as is the case of PfClpP. However, biochemical and structural data suggest that, contrary to what has been observed in other organisms, PfClpP and PfClpR do not form a stable heterocomplex in the apicoplast of P. falciparum.

Total synthesis and antibacterial testing of the A54556 cyclic acyldepsipeptides isolated from Streptomyces hawaiiensis.

The first total synthesis of all six known A54556 acyldepsipeptide (ADEP) antibiotics from Streptomyces hawaiiensis is reported. This family of compounds has a unique mechanism of antibacterial action, acting as activators of caseinolytic protease (ClpP). Assembly of the 16-membered depsipeptide core was accomplished via a pentafluorophenyl ester-based macrolactamization strategy. Late stage amine deprotection was carried out under neutral conditions by employing a mild hydrogenolysis strategy, which avoids the formation of undesired ring-opened depsipeptide side products encountered during deprotection of acid-labile protecting groups. The free amines were found to be significantly more reactive toward late stage amide bond formation as compared to the corresponding ammonium salts, giving final products in excellent yields. A thorough NMR spectroscopic analysis of these compounds was carried out to formally assign the structures and to aid with the spectroscopic assignment of ADEP analogues. The identity of two of the structures was confirmed by comparison with biologically produced samples from S. hawaiiensis. An X-ray crystallographic analysis of an ADEP analogue reveals a conformation similar to that found in cocrystal structures of ADEPs with ClpP protease. The degree of antibacterial activity of the different compounds was evaluated in vitro using MIC assays employing both Gram-positive and Gram-negative strains and a fluorescence-based biochemical assay.