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Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility. [ABSTRACT FROM AUTHOR]

Complexes of two or more proteins form many, if not most, of the intracellular "machines" that execute physical and chemical work, and transmit information. Complexes can form from stochastic post‐translational interactions of fully formed proteins, but recent attention has shifted to co‐translational interactions in which the most common mechanism involves binding of a mature constituent to an incomplete polypeptide emerging from a translating ribosome. Studies in yeast have revealed co‐translational interactions during formation of multiple major complexes, and together with recent mammalian cell studies, suggest widespread utilization of the mechanism. These translation‐dependent interactions can involve a single or multiple mRNA templates, can be uni‐ or bi‐directional, and can use multi‐protein sub‐complexes as a binding component. Here, we discuss benefits of co‐translational complex assembly including accuracy and efficiency, overcoming hidden interfaces, localized and hierarchical assembly, and reduction of orphan protein degradation, toxicity, and dominant‐negative pathogenesis, all serving to improve cell fitness. [ABSTRACT FROM AUTHOR]

Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa (P. aeruginosa, Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa. The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal b-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA. [ABSTRACT FROM AUTHOR]

Heat stress (HS) has serious negative effects on plant development and has become a major threat to agriculture. A rapid transcriptional regulatory cascade has evolved in plants in response to HS. Nuclear Factor‐Y (NF‐Y) complexes are critical for this mechanism, but how NF‐Y complexes are regulated remains unclear. In this study, we identified NF‐YC10 (NF‐Y subunit C10), a central regulator of the HS response in Arabidopsis thaliana, as a substrate of SUMOylation, an important post‐translational modification. Biochemical analysis showed that the SUMO ligase SIZ1 (SAP AND MIZ1 DOMAIN‐CONTAINING LIGASE1) interacts with NF‐YC10 and enhances its SUMOylation during HS. The SUMOylation of NF‐YC10 facilitates its interaction with and the nuclear translocation of NF‐YB3, in which the SUMO interaction motif (SIM) is essential for its efficient association with NF‐YC10. Further functional analysis indicated that the SUMOylation of NF‐YC10 and the SIM of NF‐YB3 are critical for HS‐responsive gene expression and plant thermotolerance. These findings uncover a role for the SIZ1‐mediated SUMOylation of NF‐YC10 in NF‐Y complex assembly under HS, providing new insights into the role of a post‐translational modification in regulating transcription during abiotic stress responses in plants. [ABSTRACT FROM AUTHOR]

The development of artificial protein cages has recently gained massive attention due to their promising application prospect as novel delivery vehicles for therapeutics. These nanoparticles are formed through a process called self‐assembly, in which individual subunits spontaneously arrange into highly ordered patterns via non‐covalent but specific interactions. Therefore, the first step toward the design of novel engineered protein cages is to understand the general mechanisms of their self‐assembling dynamics. Here we have developed a new computational method to tackle this problem. Our method is based on a coarse‐grained model and a diffusion–reaction simulation algorithm. Using a tetrahedral cage as test model, we showed that self‐assembly of protein cage requires of a seeding process in which specific configurations of kinetic intermediate states are identified. We further found that there is a critical concentration to trigger self‐assembly of protein cages. This critical concentration allows that cages can only be successfully assembled under a persistently high concentration. Additionally, phase diagram of self‐assembly has been constructed by systematically testing the model across a wide range of binding parameters. Finally, our simulations demonstrated the importance of protein's structural flexibility in regulating the dynamics of cage assembly. In summary, this study throws lights on the general principles underlying self‐assembly of large cage‐like protein complexes and thus provides insights to design new nanomaterials. [ABSTRACT FROM AUTHOR]

The recognition of core promoter sequences by TFIID is the first step in RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is a trilobular complex, composed of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs). Why and how TAFs are necessary for the formation of TFIID domains and how they contribute to transcription initiation remain unclear. Inducible TAF7 or TAF10 depletion, followed by comprehensive analysis of TFIID subcomplex formation, chromatin binding, and nascent transcription in mouse embryonic stem cells, result in the formation of a TAF7-lacking TFIID or a minimal core-TFIID complex, respectively. These partial complexes support TBP recruitment at promoters and nascent Pol II transcription at most genes early after depletion, but importantly, TAF10 is necessary for efficient Pol II pausing. We show that partially assembled TFIID complexes can sustain Pol II transcription initiation but cannot replace holo-TFIID over several cell divisions and/or development. [Display omitted] • TAF7 or TAF10 depletion leads to the formation of different partial TFIID complexes • Nascent Pol II transcription is not strongly affected in holo-TFIID-depleted cells • TBP is recruited at the promoter in holo-TFIID-depleted cells • Pol II pausing is affected in the absence of TAF10 Hisler et al. reveal that TAF7 and TAF10 depletion affects holo-TFIID sequential assembly, leading to the formation of partial TFIID complexes. Transcription is active and TBP is recruited at promoters when holo-TFIID is depleted. Partial TFIID complexes may sustain active transcription but cannot replace holo-TFIID over several cell divisions and/or development. [ABSTRACT FROM AUTHOR]

Coacervates are droplets formed by liquid‒liquid phase separation. An increasing number of studies have reported that coacervates play an important role in living cells, such as in the generation of membraneless organelles, and peptides contribute to condensate droplet formation. Peptides with versatile functional groups and special secondary structures, including α-helices, β-sheets and intrinsically disordered regions, provide novel insights into coacervation, such as biomimetic protocells, neurodegenerative diseases, modulations of signal transmission, and drug delivery systems. In this review, we introduce different types of peptide-based coacervates and the principles of their interactions. Additionally, we summarize the thermodynamic and kinetic mechanisms of peptide-based coacervates and the associated factors, including salt, pH, and temperature, affecting the phase separation process. We illustrate recent studies on modulating the functions of peptide-based coacervates applied in biological diseases. Finally, we propose their promising broad applications and describe the challenges of peptide-based coacervates in the future.

Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein–protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

Sideroflexins (SFXNs) comprise a family of five paralogous proteins (SFXN1-5) in metazoan species. SFXN1/2/3 function as mitochondrial serine transporters and are required for efficient mitochondrial one-carbon (1C) metabolism. SFXN4 is evolutionary divergent, and mutations in SFXN4 give rise to mitochondrial disease, pointing to a distinct function of this protein in mitochondrial biology. Using a combination of genome editing, interaction studies, and quantitative proteomics, we show that loss of SFXN4 leads to an isolated complex I assembly defect and that SFXN4 interacts with the core components of the mitochondrial complex I intermediate assembly (MCIA) complex. Our findings suggest that SFXN4 is required for the incorporation of the mtDNA-encoded ND6 subunit in the ND2 assembly module of complex I. These findings provide insights into the fundamental process of complex I assembly and functional insights into a disease-causing gene belonging to the SFXN family. [ABSTRACT FROM AUTHOR]

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA polymerase I (RNAPI) and RNAPIII are multi-heterogenic protein complexes that specialize in the transcription of highly abundant non-coding RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA). In terms of subunit number and structure, RNAPI and RNAPIII are more complex than RNAPII that synthesizes thousands of different mRNAs. Specific subunits of the yeast RNAPI and RNAPIII form associated subcomplexes that are related to parts of the RNAPII initiation factors. Prior to their delivery to the nucleus where they function, RNAP complexes are assembled at least partially in the cytoplasm. Yeast RNAPI and RNAPIII share heterodimer Rpc40-Rpc19, a functional equivalent to the αα homodimer which initiates assembly of prokaryotic RNAP. In the process of yeast RNAPI and RNAPIII biogenesis, Rpc40 and Rpc19 form the assembly platform together with two small, bona fide eukaryotic subunits, Rpb10 and Rpb12. We propose that this assembly platform is co-translationally seeded while the Rpb10 subunit is synthesized by cytoplasmic ribosome machinery. The translation of Rpb10 is stimulated by Rbs1 protein, which binds to the 3′-untranslated region of RPB10 mRNA and hypothetically brings together Rpc19 and Rpc40 subunits to form the αα-like heterodimer. We suggest that such a co-translational mechanism is involved in the assembly of RNAPI and RNAPIII complexes.

Rotor ATPases are large multisubunit membrane protein complexes found in all kingdoms of life. The membrane parts of these ATPases include a ring-like assembly, so-called c-ring, consisting of several subunits c, plugged by a patch of phospholipids. In this report, we use a nature-inspired approach to model the assembly of the spinach (Spinacia oleracea) c14 ring protein-lipid complex, where partially assembled oligomers are pulled toward each other using a biasing potential. The resulting assemblies contain 23 to 26 encapsulated plug lipids, general position of which corresponds well to experimental maps. However, best fit to experimental data is achieved with 15 to 17 lipids inside the c-ring. In all of the simulations, the lipids from one leaflet (loop side of the c subunit) are ordered and static, whereas the lipids from the other leaflet are disordered and dynamic. Spontaneous permeation of water molecules toward Glu61 at the active site is also observed. The presented assembly approach is expected to be generalizable to other protein complexes with encapsulated lipid patches.

Ligand binding site structure has profound consequences for the evolution of function of protein complexes, particularly in homomers—complexes comprising multiple copies of the same protein. Previously, we have shown that homomers with multichain binding sites (MBSs) are characterized by more conserved binding sites and quaternary structure, and qualitatively different allosteric pathways than homomers with single-chain binding sites (SBSs) or monomers. Here, using computational methods, we show that the folds of single-domain MBS and SBS homomers are different, and SBS homomers are likely to be folded cotranslationally, while MBS homomers are more likely to form post-translationally and rely on more advanced folding-assistance and quality control mechanisms, which include chaperonins. In addition, our findings demonstrate that MBS homomers are qualitatively different from monomers, while SBS homomers are much less distinct, supporting the hypothesis that the evolution of quaternary structure in SBS homomers is significantly influenced by stochastic processes. Unlabelled Image [ABSTRACT FROM AUTHOR]

Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems either promote the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. Although well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially encoded proteins, key subunits of the oxidative phosphorylation (OXPHOS) system. Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial protein biogenesis and quality control. Conserved prohibitin (PHB)/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in the vicinity of the mitoribosomal tunnel exit allows for a prompt decision on whether newly synthesized proteins are fed into OXPHOS assembly or are degraded. [Display omitted] • Fate decisions of mitochondrially encoded proteins are achieved at mitoribosomes • These decisions directly occur upon the completion of protein synthesis • Thereby, OXPHOS assembly factors colocalize with the PHB/m-AAA protease complex • If OXPHOS assembly is delayed, nascent proteins are degraded via the PHB/m-AAA Dedicated protein quality control systems ensure the folding of newly synthesized proteins at ribosomes. Kohler et al. reveal such a system for mitochondrially encoded proteins in the form of the PHB/m-AAA protease associated with mitoribosomes. Thereby, interactions with OXPHOS assembly factors or the PHB/m-AAA protease determine the fate of nascent proteins. [ABSTRACT FROM AUTHOR]

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes. [Display omitted] • TRiC assembles hierarchically through defined intermediates along a biochemical axis • Subcomplexes of adjacent subunits CCT2-4-5-7 nucleate concurrent double-ring assembly • Monomeric CCT8 and labile CCT1, CCT3, and CCT6 join individually to form final complex • Hierarchical regulation disfavors alternate CCT arrangements with aberrant function Moreira et al. show that TRiC/CCT chaperonin assembly involves a hierarchical pathway of intermediates segregating subunit properties along a biochemical axis with subunits CCT2, CCT4, CCT5, and CCT7 nucleating assembly while CCT8, CCT1, CCT3, and CCT6 join the ring individually. Unassembled oligomers and subunits are degraded by proteasomal and lysosomal pathways, preventing the assembly of noncanonical chaperonin arrangements with aberrant functions. [ABSTRACT FROM AUTHOR]

Encapsulin is a class of nanocompartments that is unique in bacteria and archaea to confine enzymatic activities and sequester toxic reaction products. Here we present a 2.87 Å resolution cryo-EM structure of Thermotoga maritima encapsulin with heterologous protein complex loaded. It is the first successful case of expressing encapsulin and heterologous cargo protein in the insect cell system. Although we failed to reconstruct the cargo protein complex structure due to the signal interference of the capsid shell, we were able to observe some unique features of the cargo-loaded encapsulin shell, for example, an extra density at the fivefold pore that has not been reported before. These results would lead to a more complete understanding of the encapsulin cargo assembly process of T. maritima.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249 -encoded transmembrane (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.

Mitochondrial genome has undergone significant reduction in a course of evolution; however, it still contains a set of proteinencoding genes and requires translational machinery for their expression. Mitochondrial translation is of the prokaryotic type with several remarkable differences. This review is dedicated to one of the most puzzling features of mito-chondrial protein synthesis, namely, the system of translational activators, i.e., proteins that specifically regulate translation of individual mitochondrial mRNAs and couple protein biosynthesis with the assembly of mitochondrial respiratory chain complexes. The review does not claim to be a comprehensive analysis of all published data; it is rather focused on the idea of the "core component" of the translational activator system. [ABSTRACT FROM AUTHOR]

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 ( p article for a rrangement of q uaternary structure) to more accurately represent its unique function. [ABSTRACT FROM AUTHOR]

Background: Large sets of protein-protein interaction data coming either from biological experiments or predictive methods are available and can be combined to construct networks from which information about various cell processes can be extracted. We have developed an in silico approach based on these information to model the biogenesis of multiprotein complexes in the yeast Saccharomyces cerevisiae. Results: Firstly, we have built three protein interaction networks by collecting the protein-protein interactions, which involved the subunits of three complexes, from different databases. The protein-protein interactions come from different kinds of biological experiments or are predicted. We have chosen the elongator and the mediator head complexes that are soluble and exhibit an architecture with subcomplexes that could be functional modules, and the mitochondrial bc1 complex, which is an integral membrane complex and for which a late assembly subcomplex has been described. Secondly, by applying a clustering strategy to these networks, we were able to identify subcomplexes involved in the biogenesis of the complexes as well as the proteins interacting with each subcomplex. Thirdly, in order to validate our in silico results for the cytochrome bc1 complex we have analysed the physical interactions existing between three subunits by performing immunoprecipitation experiments in several genetic context. Conclusions: For the two soluble complexes (the elongator and mediator head), our model shows a strong clustering of subunits that belong to a known subcomplex or module. For the membrane bc1 complex, our approach has suggested new interactions between subunits in the early steps of the assembly pathway that were experimentally confirmed. Scripts can be downloaded from the site: http://bim.igmors.u-psud.fr/isips. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans. These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme. IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation. [ABSTRACT FROM AUTHOR]

Holliday junction (HJ) is a four-way structured DNA intermediate in homologous recombination. In bacteria, the HJ-specific binding protein RuvA and the motor protein RuvB together form the RuvAB complex to catalyze HJ branch migration. Pseudomonas aeruginosa ( P. aeruginosa , Pa) is a ubiquitous opportunistic bacterial pathogen that can cause serious infection in a variety of host species, including vertebrate animals, insects and plants. Here, we describe the cryo-Electron Microscopy (cryo-EM) structure of the RuvAB-HJ intermediate complex from P. aeruginosa . The structure shows that two RuvA tetramers sandwich HJ at the junction center and disrupt base pairs at the branch points of RuvB-free HJ arms. Eight RuvB subunits are recruited by the RuvA octameric core and form two open-rings to encircle two opposite HJ arms. Each RuvB subunit individually binds a RuvA domain III. The four RuvB subunits within the ring display distinct subdomain conformations, and two of them engage the central DNA duplex at both strands with their C-terminal β-hairpins. Together with the biochemical analyses, our structure implicates a potential mechanism of RuvB motor assembly onto HJ DNA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Zhou, Dai, Chao, Liu, Huang, Qu and Lin.)

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo . Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function. [ABSTRACT FROM AUTHOR]

Costly complex assembly operations supported by augmented reality demands endeavors to achieve a comparative and experimental depiction of different circumstances for assays to analyze fluctuating conclusions where the situation with limited number of tries has compelled to recognize that variations exist depending on test conditions. Then developments in emergent technologies, for training in Maintenance and Repair Operations (MRO), must be evaluated. Scenarios are focused on assessing an AR request, by stating a confrontation of declarations about assembly time and comparisons respect to outstanding authors who conduct assembly operations. An assembly operations case study establishes a small sample size during experimentation. [ABSTRACT FROM AUTHOR]

The development of artificial protein cages has recently gained massive attention due to their promising application prospect as novel delivery vehicles for therapeutics. These nanoparticles are formed through a process called self-assembly, in which individual subunits spontaneously arrange into highly ordered patterns via non-covalent but specific interactions. Therefore, the first step toward the design of novel engineered protein cages is to understand the general mechanisms of their self-assembling dynamics. Here we have developed a new computational method to tackle this problem. Our method is based on a coarse-grained model and a diffusion-reaction simulation algorithm. Using a tetrahedral cage as test model, we showed that self-assembly of protein cage requires of a seeding process in which specific configurations of kinetic intermediate states are identified. We further found that there is a critical concentration to trigger self-assembly of protein cages. This critical concentration allows that cages can only be successfully assembled under a persistently high concentration. Additionally, phase diagram of self-assembly has been constructed by systematically testing the model across a wide range of binding parameters. Finally, our simulations demonstrated the importance of protein's structural flexibility in regulating the dynamics of cage assembly. In summary, this study throws lights on the general principles underlying self-assembly of large cage-like protein complexes and thus provides insights to design new nanomaterials., (© 2022 The Protein Society.)

Coacervates are droplets formed by liquid‒liquid phase separation. An increasing number of studies have reported that coacervates play an important role in living cells, such as in the generation of membraneless organelles, and peptides contribute to condensate droplet formation. Peptides with versatile functional groups and special secondary structures, including α-helices, β-sheets and intrinsically disordered regions, provide novel insights into coacervation, such as biomimetic protocells, neurodegenerative diseases, modulations of signal transmission, and drug delivery systems. In this review, we introduce different types of peptide-based coacervates and the principles of their interactions. Additionally, we summarize the thermodynamic and kinetic mechanisms of peptide-based coacervates and the associated factors, including salt, pH, and temperature, affecting the phase separation process. We illustrate recent studies on modulating the functions of peptide-based coacervates applied in biological diseases. Finally, we propose their promising broad applications and describe the challenges of peptide-based coacervates in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ma, Fang and Wang.)

The NLRP3 inflammasome is a critical component of innate immunity that activates caspase-1 to induce inflammation in response to a wide spectrum of endogenous and exogenous stimuli. NLRP3 inflammasome activation has been shown by assays for the cleavage of caspase-1 and gasdermin D, the maturation of IL-1β and IL-18, and ASC speck formation in innate immune cells such as macrophages and monocytes. Recently, NEK7 has been revealed as an essential regulator for NLRP3 inflammasome activation by forming high-molecular-weight complexes with NLRP3. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to study multi-protein complexes in many experimental systems. Here, we provide a detailed protocol to detect NLRP3 inflammasome activation and NLRP3-NEK7 complex assembly in mouse macrophages by Western blot and BN-PAGE., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The evolutionarily conserved Elongator complex, which is composed of six subunits elongator protein 1 (Elp1 to -6), plays vital roles in gene regulation. The molecular hallmark of familial dysautonomia (FD) is the splicing mutation of Elp1 [also known as IκB kinase complex-associated protein (IKAP)] in the nervous system that is believed to be the primary cause of the devastating symptoms of this disease. Here, we demonstrate that disease-related mutations in Elp1 affect Elongator assembly, and we have determined the structure of the C-terminal portion of human Elp1 (Elp1-CT), which is sufficient for full-length Elp1 dimerization, as well as the structure of the cognate dimerization domain of yeast Elp1 (yElp1-DD). Our study reveals that the formation of the Elp1 dimer contributes to its stability in vitro and in vivo and is required for the assembly of both the human and yeast Elongator complexes. Functional studies suggest that Elp1 dimerization is essential for yeast viability. Collectively, our results identify the evolutionarily conserved dimerization domain of Elp1 and suggest that the pathological mechanisms underlying the onset and progression of Elp1 mutation-related disease may result from impaired Elongator activities. [ABSTRACT FROM AUTHOR]

Phycocyanin is one of the two phycobiliproteins always found in the Phycobilisome antenna complex. It is always situated at the ends of the peripheral rods, adjacent to the core cylinders composed of allophycocyanin. The basic phycocyanin monomer is an (αβ) dimer of globin-like subunits with three covalently linked phycocyanobilin cofactors. Monomers assemble further into trimers, hexamers, and rods which include non-pigmented linker proteins. Upon isolation in low ionic strength solution, rods quickly disintegrate into phycocyanin trimers, which lose contacts with other phycobiliproteins and with the linker proteins. The trimers, however, are quite stable and only the presence of high concentrations of chaotropic agents (such as urea), very acidic solutions, or elevated temperatures induces monomerization, followed by separation between the subunits. We have recently determined the crystal structures of phycocyanin from the thremophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 or 4 M urea, and shown that 4 M urea monomerizes the phycocyanin trimers. In this paper, we will describe the phycocyanin structures in 2 and 4 M urea more completely. By mapping out the urea positions, we describe the structural elements within the trimeric interaction interface that may be interrupted by the presence of 4 M urea. In addition, we also identify what are the structural characteristics that prevent 4 M urea from inducing subunit dissociation. [ABSTRACT FROM AUTHOR]

Abstract: The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2–5 cylinders surrounded by 6–8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo. [Copyright &y& Elsevier]

The process of troubleshooting an aircraft engine requires highly skilled and trained personnel who must be able to respond effectively to any circumstance; therefore, new methods of training to accelerate the cognitive processes of technicians must be integrated in the industry. In this matter the Augmented Reality technology represents an innovative tool that can ensure the efficient and correct transfer of knowledge. The numbers of errors during maintenance tasks can be reduced, AR provides information that is generally not easily available during maintenance operations because, in general, the troubleshooting process for airplane engine is a highly complex task and the diagnosis of a failure is critical for the passengers’ safety. This research focuses on training and execution of tasks where an aviation technician must be familiarized with a wide variety of technical data, physical components of mechanical systems and the regulations that must be followed to release an airplane for flight, the specialist must develop a correct mind map of the system and should be able to troubleshoot if necessary. The case of study is the 737 Engine Bleed Air System that is designed to provide engine compressed air to air conditioning pack with the purpose of air pressurization during flight; engine air from the compressor is used, from the 5° and the 9° stage in a safe an economical way, knowledge of the correct function of the components will increase safety and considerably reduce cost of maintenance operations. The purpose of the investigation was to develop an ergonomic tool than improves the cognitive process of technician during training for the troubleshooting techniques of the aircraft, but it also can be used to the everyday task by capturing the know-how and helpful tips from more experienced operators. A mobile solution that functions on regular tablets was delivered to enhance the troubleshooting techniques and maintenance procedures of the Engine Air Bleed System, the software can function on two aspects for training and in situ operations. A commercial aeronautical training kit was used to validate the Fault Isolation Software; the results showed that the augmented reality technique takes 17% less time and a quality increment of 24% for this complex assembly system. [ABSTRACT FROM AUTHOR]

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.

• Rbs1 is mRNA-binding protein that participates in RNAP III assembly. • Early step of RNAP III assembly involves formation of Rpb10-Rpc19-Rpc40 heterotrimer. • Co-translational mechanism of RNAP III assembly is proposed. Detailed knowledge of structures of yeast RNA polymerases (RNAPs) contrasts with the limited information that is available on the control of their assembly. RNAP enzymes are large heteromeric complexes that function in the nucleus, but they are assembled in the cytoplasm and imported to the nucleus with help from specific auxiliary factors. Here, I review a recent study that suggests that the formation of an early-stage assembly intermediate of the RNAP III complex occurs through a co-translational mechanism. According to our hypothesis, RNAP III assembly might be seeded while the Rpb10 subunit of the enzyme core is being synthesized by cytoplasmic ribosome machinery. The co-translational assembly of RNAP III is mediated by Rbs1 protein which binds to 3′-untranslated regions in mRNA in a way that depends on the R3H domain in the Rbs1 sequence. [ABSTRACT FROM AUTHOR]

Bacterial cellulosomes are generally believed to assemble at random, like those produced by Clostridium cellulolyticum. They are composed of one scaffolding protein bearing eight homologous type I cohesins that bind to any of the type I dockerins borne by the 62 cellulosomal subunits, thus generating highly heterogeneous complexes. In the present study, the heterogeneity and random assembly of the cellulosomes were evaluated with a simpler model: a miniscaffoldin containing three C. cellulolyticum cohesins and three cellulases of the same bacterium bearing the cognate dockerin (Cel5A, Cel48F, and Cel9G). Surprisingly, rather than the expected randomized integration of enzymes, the assembly of the minicellulosome generated only three distinct types of complex out of the 10 possible combinations, thus indicating preferential integration of enzymes upon binding to the scaffoldin. A hybrid scaffoldin that displays one cohesin from C. cellulolyticum and one from C. thermocellum, thus allowing sequential integration of enzymes, was exploited to further characterize this phenomenon. The initial binding of a given enzyme to the C. thermocellum cohesin was found to influence the type of enzyme that subsequently bound to the C. cellulolyticum cohesin. The preferential integration appears to be related to the length of the inter-cohesin linker. The data indicate that the binding of a cellulosomal enzyme to a cohesin has a direct influence on the dockerin-bearing proteins that will subsequently interact with adjacent cohesins. Thus, despite the general lack of specificity of the cohesin-dockerin interaction within a given species and type, bacterial cellulosomes are not necessarily assembled at random. [ABSTRACT FROM AUTHOR]

Abstract: X-ray crystal structures of the isolated phycobiliprotein components of the phycobilisome have provided high resolution details to the description of this light harvesting complex at different levels of complexity and detail. The linker-independent assembly of trimers into hexamers in crystal lattices of previously determined structures has been observed in almost all of the phycocyanin (PC) and allophycocyanin (APC) structures available in the Protein Data Bank. In this paper we describe the X-ray crystal structures of PC and APC from Synechococcus elongatus sp. PCC 7942, PC from Synechocystis sp. PCC 6803 and PC from Thermosynechococcus vulcanus crystallized in the presence of urea. All five structures are highly similar to other PC and APC structures on the levels of subunits, monomers and trimers. The Synechococcus APC forms a unique loose hexamer that may show the structural requirements for core assembly and rod attachment. While the Synechococcus PC assembles into the canonical hexamer, it does not further assemble into rods. Unlike most PC structures, the Synechocystis PC fails to form hexamers. Addition of low concentrations of urea to T. vulcanus PC inhibits this proteins propensity to form hexamers, resulting in a crystal lattice composed of trimers. The molecular source of these differences in assembly and their relevance to the phycobilisome structure is discussed. [Copyright &y& Elsevier]

Chloroplasts are the endosymbiotic descendants of cyanobacterium-like prokaryotes. Present genomes of plant and green algae chloroplasts (plastomes) contain ~100 genes mainly encoding for their transcription-/translation-machinery, subunits of the thylakoid membrane complexes (photosystems II and I, cytochrome b f, ATP synthase), and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Nevertheless, proteomic studies have identified several thousand proteins in chloroplasts indicating that the majority of the plastid proteome is not encoded by the plastome. Indeed, plastid and host cell genomes have been massively rearranged in the course of their co-evolution, mainly through gene loss, horizontal gene transfer from the cyanobacterium/chloroplast to the nucleus of the host cell, and the emergence of new nuclear genes. Besides structural components of thylakoid membrane complexes and other (enzymatic) complexes, the nucleus provides essential factors that are involved in a variety of processes inside the chloroplast, like gene expression (transcription, RNA-maturation and translation), complex assembly, and protein import. Here, we provide an overview on regulatory factors that have been described and characterized in the past years, putting emphasis on mechanisms regulating the expression and assembly of the photosynthetic thylakoid membrane complexes. [ABSTRACT FROM AUTHOR]

The de novo assembly of photosystem II (PSII) depends on a variety of assisting factors. We have previously shown that two of them, namely, YCF48 and Sll0933, mutually interact and form a complex (Rengstl et al. in J Biol Chem 286:21944-21951, ). To gain further insights into the importance of the YCF48/Sll0933 interaction, an ycf48 sll0933 double mutant was constructed and its phenotype was compared with the single mutants' phenotypes. Analysis of fluorescence spectra and oxygen evolution revealed high-light sensitivity not only for YCF48 deficient strains but also for sll0933, which, in addition, showed reduced synthesis and accumulation of newly synthesized CP43 and CP47 proteins in pulse-labeling experiments. In general, the phenotypic characteristics of ycf48 sll0933 were dominated by the effect of the ycf48 deletion and additional inactivation of the sll0933 gene showed only negligible additional impairments with regard to growth, absorption spectra and accumulation of PSII-related proteins and assembly complexes. In yeast split-ubiquitin analyses, the interaction between YCF48 and Sll0933 was confirmed and, furthermore, support for direct binding of Sll0933 to CP43 and CP47 was obtained. Our data provide important new information which further refines our knowledge about the PSII assembly process and role of accessory protein factors within it. [ABSTRACT FROM AUTHOR]

Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein-protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

The primary function of mitochondria is respiration, where catabolism of substrates is coupled to ATP synthesis via oxidative phosphorylation. In plants, mitochondrial composition is relatively complex and flexible and has specific pathways to support photosynthetic processes in illuminated leaves. This review begins with outlining current models of mitochondrial composition in plant cells, with an emphasis upon the assembly of the complexes of the classical electron transport chain (ETC). Next, we focus upon the comparative analysis of mitochondrial function from different tissue types. A prominent theme in the plant mitochondrial literature involves linking mitochondrial composition to environmental stress responses, and this review then gives a detailed outline of how oxidative stress impacts upon the plant mitochondrial proteome with particular attention to the role of transition metals. This is followed by an analysis of the signaling capacity of mitochondrial reactive oxygen species, which studies the transcriptional changes of stress responsive genes as a framework to define specific signals emanating from the mitochondrion. Finally, specific mitochondrial roles during exposure to harsh environments are outlined, with attention paid to mitochondrial delivery of energy and intermediates, mitochondrial support for photosynthesis, and mitochondrial processes operating within root cells that mediate tolerance to anoxia and unfavorable soil chemistries. [ABSTRACT FROM AUTHOR]

Abstract: Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90). [Copyright &y& Elsevier]