RESUMO
Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains.
Assuntos
Sistemas CRISPR-Cas , Conjugação Genética , Klebsiella pneumoniae , Plasmídeos , Plasmídeos/genética , Klebsiella pneumoniae/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transferência Genética Horizontal , Bacteriófagos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Bases de Dados Genéticas , Endodesoxirribonucleases , Sistemas CRISPR-Cas/genética , Filogenia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/classificação , Proteínas Associadas a CRISPR/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/classificação , Endodesoxirribonucleases/genética , Enciclopédias como AssuntoRESUMO
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Edição de Genes , Arabidopsis/genética , Sistemas CRISPR-Cas , Plantas/genética , Genoma de Planta/genética , Fatores de Transcrição/genética , Proteínas de Homeodomínio/genética , Proteínas de Arabidopsis/genéticaRESUMO
CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.
Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Animais , Humanos , Microscopia Crioeletrônica , Edição de Genes , Genoma , Bacteriófagos/genética , DNA , RNA , Mamíferos/genéticaRESUMO
CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.
Assuntos
Antitoxinas , Edição de Genes , Antitoxinas/genética , Bactérias , Biologia , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis , RNA Bacteriano , RetroelementosRESUMO
CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Clivagem do DNA , Conformação Molecular , Bacteriófagos/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Técnicas Genéticas , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Proteínas de Bactérias/química , Biocatálise , Cristalografia por Raios X , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
CRISPR-Cas systems are found widely in prokaryotes, where they provide adaptive immunity against virus infection and plasmid transformation. We describe a minimal functional CRISPR-Cas system, comprising a single ~70-kilodalton protein, CasΦ, and a CRISPR array, encoded exclusively in the genomes of huge bacteriophages. CasΦ uses a single active site for both CRISPR RNA (crRNA) processing and crRNA-guided DNA cutting to target foreign nucleic acids. This hypercompact system is active in vitro and in human and plant cells with expanded target recognition capabilities relative to other CRISPR-Cas proteins. Useful for genome editing and DNA detection but with a molecular weight half that of Cas9 and Cas12a genome-editing enzymes, CasΦ offers advantages for cellular delivery that expand the genome editing toolbox.
Assuntos
Bacteriófagos/genética , Sistemas CRISPR-Cas , Edição de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente EspaçadasRESUMO
In the version of this Article originally published, author Carolina Falcón Garcia's name was coded wrongly, resulting in it being incorrect when exported to citation databases. This has now been corrected, though no visible changes will be apparent.
RESUMO
Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/genética , Rhodocyclaceae/genética , Sequência de Bases , Espectrometria de Massas , Microscopia Eletrônica , Plasmídeos/genética , Análise de Sequência de RNARESUMO
Adaptive immunity of prokaryotes is mediated by CRISPR-Cas systems that employ a large variety of Cas protein effectors to identify and destroy foreign genetic material. The different targeting mechanisms of Cas proteins rely on the proper protection of the host genome sequence while allowing for efficient detection of target sequences, termed protospacers. A short DNA sequence, the protospacer-adjacent motif (PAM), is frequently used to mark proper target sites. Cas proteins have evolved a multitude of PAM-interacting domains, which enables them to cope with viral anti-CRISPR measures that alter the sequence or accessibility of PAM elements. In this review, we summarize known PAM recognition strategies for all CRISPR-Cas types. Available structures of target bound Cas protein effector complexes highlight the diversity of mechanisms and domain architectures that are employed to guarantee target specificity.
Assuntos
Sistemas CRISPR-Cas/genética , Motivos de Nucleotídeos/genética , Adaptação Fisiológica/genética , Autoimunidade/genética , Sequência de Bases , Modelos Moleculares , Ribonucleases/metabolismoRESUMO
BACKGROUND: Selection for a certain trait in microbes depends on the genetic background of the strain and the selection pressure of the environmental conditions acting on the cells. In contrast to the sessile state in the biofilm, various bacterial cells employ flagellum-dependent motility under planktonic conditions suggesting that the two phenotypes are mutually exclusive. However, flagellum dependent motility facilitates the prompt establishment of floating biofilms on the air-medium interface, called pellicles. Previously, pellicles of B. subtilis were shown to be preferably established by motile cells, causing a reduced fitness of non-motile derivatives in the presence of the wild type strain. RESULTS: Here, we show that lack of active flagella promotes the evolution of matrix overproducers that can be distinguished by the characteristic wrinkled colony morphotype. The wrinkly phenotype is associated with amino acid substitutions in the master repressor of biofilm-related genes, SinR. By analyzing one of the mutations, we show that it alters the tetramerization and DNA binding properties of SinR, allowing an increased expression of the operon responsible for exopolysaccharide production. Finally, we demonstrate that the wrinkly phenotype is advantageous when cells lack flagella, but not in the wild type background. CONCLUSIONS: Our experiments suggest that loss of function phenotypes could expose rapid evolutionary adaptation in bacterial biofilms that is otherwise not evident in the wild type strains.
Assuntos
Bacillus subtilis/fisiologia , Evolução Biológica , Bacillus subtilis/citologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Movimento , Mutação/genética , Taxa de Mutação , Regiões Operadoras Genéticas/genética , Óperon , Fenótipo , Seleção GenéticaRESUMO
Efficient adaptation to environmental changes is pivotal for all bacterial cells. Almost all bacterial species depend on the conserved stringent response system to prompt timely transcriptional and metabolic responses according to stress conditions and nutrient depletion. The stringent response relies on the stress-dependent synthesis of the second messenger nucleotides and alarmones (p)ppGpp, which pleiotropically target and reprogram processes that consume cellular resources, such as ribosome biogenesis. Here we show that (p)ppGpp acts on the ribosome biogenesis GTPase A (RbgA) of Gram-positive bacteria. Using X-ray crystallography, hydrogen-deuterium exchange MS (HDX-MS) and kinetic analysis, we demonstrate that the alarmones (p)ppGpp bind to RbgA in a manner similar to that of binding by GDP and GTP and thereby act as competitive inhibitors. Our structural analysis of Staphylococcus aureus RbgA bound to ppGpp and pppGpp at 1.8 and 1.65 Å resolution, respectively, suggested that the alarmones (p)ppGpp prevent the active GTPase conformation of RbgA by sterically blocking the association of its G2 motif via their 3'-pyrophosphate moieties. Taken together, our structural and biochemical characterization of RbgA in the context of the alarmone-mediated stringent response reveals how (p)ppGpp affects the function of RbgA and reprograms this GTPase to arrest the ribosomal large subunit.
Assuntos
Inibidores Enzimáticos/farmacologia , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/química , Guanosina Pentafosfato/farmacologia , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Cristalografia por Raios X , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Magnésio/metabolismo , Modelos Moleculares , Domínios Proteicos , Staphylococcus aureus/enzimologiaRESUMO
Closely related microorganisms often cooperate, but the prevalence and stability of cooperation between different genotypes remain debatable. Here, we track the evolution of pellicle biofilms formed through genetic division of labour and ask whether partially deficient partners can evolve autonomy. Pellicles of Bacillus subtilis rely on an extracellular matrix composed of exopolysaccharide (EPS) and the fibre protein TasA. In monocultures, ∆eps and ∆tasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture. Interestingly, cooperation collapses on an evolutionary timescale and ∆tasA gradually outcompetes its partner ∆eps. Pellicle formation can evolve independently from division of labour in ∆eps and ∆tasA monocultures, by selection acting on the residual matrix component, TasA or EPS, respectively. Using a set of interdisciplinary tools, we unravel that the TasA producer (∆eps) evolves via an unconventional but reproducible substitution in TasA that modulates the biochemical properties of the protein. Conversely, the EPS producer (ΔtasA) undergoes genetically variable adaptations, all leading to enhanced EPS secretion and biofilms with different biomechanical properties. Finally, we revisit the collapse of division of labour between Δeps and ΔtasA in light of a strong frequency versus exploitability trade-off that manifested in the solitarily evolving partners. We propose that such trade-off differences may represent an additional barrier to evolution of division of labour between genetically distinct microorganisms.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Divisão Celular/fisiologia , Adaptação Fisiológica , Amiloide/química , Amiloide/ultraestrutura , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Técnicas de Cocultura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Mutação , Fenótipo , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Multimerização ProteicaRESUMO
CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/metabolismo , Endonucleases/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Cristalografia por Raios X , DNA/química , DNA/genética , Endonucleases/química , Endonucleases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ligação Proteica , Conformação Proteica , Capuzes de RNA/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Shewanella putrefaciens/enzimologia , Shewanella putrefaciens/genética , Relação Estrutura-AtividadeRESUMO
Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli/genética , Shewanella putrefaciens/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Bacteriófago lambda/fisiologia , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Escherichia coli/imunologia , Escherichia coli/metabolismo , Escherichia coli/virologia , Expressão Gênica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Shewanella putrefaciens/imunologia , Shewanella putrefaciens/metabolismo , Shewanella putrefaciens/virologia , Transformação BacterianaRESUMO
Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus.
Assuntos
Chaperonas Moleculares/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Núcleo Celular/genética , Chaperonas Moleculares/metabolismo , RNA Ribossômico/genética , Ribossomos/metabolismo , Saccharomyces cerevisiaeRESUMO
Regulated turnover of integrin receptors is essential for cell adhesion and migration. Pathways selectively regulating ß1-integrin recycling are implicated in cancer invasion and metastasis, yet proteins required for the internalization of this pro-invasive integrin remain to be identified. Here, we uncover formin-like 2 (FMNL2) as a critical regulator of ß1-integrin internalization downstream of protein kinase C (PKC). PKCα associates with and phosphorylates FMNL2 at S1072 within its Diaphanous autoregulatory region, leading to the release of formin autoinhibition. Phosphorylation of FMNL2 triggers its rapid relocation and promotes its interaction with the cytoplasmic tails of the α-integrin subunits for ß1-integrin endocytosis. FMNL2 drives ß1-integrin internalization and invasive motility in a phosphorylation-dependent manner, while a FMNL2 mutant defective in actin assembly interferes with ß1-integrin endocytosis and cancer cell invasion. Our data establish a role for FMNL2 in the regulation of ß1-integrin and provide a mechanistic understanding of the function of FMNL2 in cancer invasiveness.
Assuntos
Movimento Celular , Integrina beta1/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Citoplasma/metabolismo , Endocitose , Endossomos/metabolismo , Ativação Enzimática , Forminas , Células HEK293 , Células HeLa , Humanos , Integrina beta1/química , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Invasividade Neoplásica , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas/químicaRESUMO
Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat ß-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.
Assuntos
Proteínas Fúngicas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
The bacterial flagellum is a motility structure and represents one of the most sophisticated nanomachines in the biosphere. Here, we review the current knowledge on the flagellum, its architecture with respect to differences between Gram-negative and Gram-positive bacteria and other species-specific variations (e.g. the flagellar filament protein, Flagellin). We further focus on the mechanism by which the two nucleotide-binding proteins FlhF and FlhG ensure the correct reproduction of flagella place and number (the flagellation pattern). We will finish the review with an overview of current biotechnological applications, and a perspective of how understanding flagella can contribute to developing modules for synthetic approaches.