Encapsulins Giessen, Tobias W
Annual review of biochemistry,
06/2022, Letnik:
91, Številka:
1
Journal Article
Recenzirano
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Subcellular compartmentalization is a defining feature of all cells. In prokaryotes, compartmentalization is generally achieved via protein-based strategies. The two main classes of microbial protein ...compartments are bacterial microcompartments and encapsulin nanocompartments. Encapsulins self-assemble into proteinaceous shells with diameters between 24 and 42 nm and are defined by the viral HK97-fold of their shell protein. Encapsulins have the ability to encapsulate dedicated cargo proteins, including ferritin-like proteins, peroxidases, and desulfurases. Encapsulation is mediated by targeting sequences present in all cargo proteins. Encapsulins are found in many bacterial and archaeal phyla and have been suggested to play roles in iron storage, stress resistance, sulfur metabolism, and natural product biosynthesis. Phylogenetic analyses indicate that they share a common ancestor with viral capsid proteins. Many pathogens encode encapsulins, and recent evidence suggests that they may contribute toward pathogenicity. The existing information on encapsulin structure, biochemistry, biological function, and biomedical relevance is reviewed here.
•Encapsulins are a new class of microbial protein nanocompartments with diameters of 24 or 32nm.•They naturally encapsulate a variety of cargo proteins related to oxidative stress response.•Cargo ...proteins are directed to the interior of encapsulin cages by C-terminal tags.•Encapsulins can be used as cell-specific optical probes and targeted delivery systems.•Non-native enzymes can be encapsulated within encapsulins in vivo and retain their activity.
Compartmentalization is one of the defining features of life. Cells use protein compartments to exert spatial control over their metabolism, store nutrients and create unique microenvironments needed for essential physiological processes. Encapsulins are a recently discovered class of protein nanocompartments found in bacteria and archaea that naturally encapsulate cargo proteins. A short C-terminal targeting sequence directs the highly specific encapsulation process in vivo. Here, I will initially discuss the properties, diversity and putative function of encapsulins. The unique characteristics and potential uses of the self-sorting cargo-packaging process found in encapsulin systems will then be highlighted. Examples for the application of encapsulins as cell-specific optical nanoprobes and targeted therapeutic delivery systems will be discussed with an emphasis on the ability to integrate multiple functionalities within a single nanodevice. By fusing targeting sequences to non-native proteins, encapsulins can also be used as specific nanocontainers and enzymatic nanoreactors in vivo. I will end by briefly discussing future avenues for encapsulin research related to both basic microbial metabolism and applications in biomedicine, catalysis and materials science.
Bacterial protein compartments concentrate and sequester enzymes, thereby regulating biochemical reactions. Here, we generated a new functional nanocompartment in Escherichia coli by engineering the ...MS2 phage capsid protein to encapsulate multiple cargo proteins. Sequestration of multiple proteins in MS2-based capsids was achieved by SpyTag/SpyCatcher protein fusions that covalently crosslinked with the interior surface of the capsid. Further, the functional two-enzyme indigo biosynthetic pathway could be targeted to the engineered capsids, leading to a 60 % increase in indigo production in vivo. The enzyme-loaded particles could be purified in their active form and showed enhanced long-term stability in vitro (about 95 % activity after seven days) compared with free enzymes (about 5 % activity after seven days). In summary, this engineered in vivo encapsulation system provides a simple and versatile way for generating highly stable multi-enzyme nanoreactors for in vivo and in vitro applications.
Encapsulins are a class of microbial protein compartments defined by the viral HK97-fold of their capsid protein, self-assembly into icosahedral shells, and dedicated cargo loading mechanism for ...sequestering specific enzymes. Encapsulins are often misannotated and traditional sequence-based searches yield many false positive hits in the form of phage capsids. Here, we develop an integrated search strategy to carry out a large-scale computational analysis of prokaryotic genomes with the goal of discovering an exhaustive and curated set of all HK97-fold encapsulin-like systems. We find over 6,000 encapsulin-like systems in 31 bacterial and four archaeal phyla, including two novel encapsulin families. We formulate hypotheses about their potential biological functions and biomedical relevance, which range from natural product biosynthesis and stress resistance to carbon metabolism and anaerobic hydrogen production. An evolutionary analysis of encapsulins and related HK97-type virus families shows that they share a common ancestor, and we conclude that encapsulins likely evolved from HK97-type bacteriophages.
Rhodanese‐like domains (RLDs) represent a widespread protein family canonically involved in sulfur transfer reactions between diverse donor and acceptor molecules. RLDs mediate these transsulfuration ...reactions via a transient persulfide intermediate, created by modifying a conserved cysteine residue in their active sites. RLDs are involved in various aspects of sulfur metabolism, including sulfide oxidation in mitochondria, iron–sulfur cluster biogenesis, and thio‐cofactor biosynthesis. However, due to the inherent complexity of sulfur metabolism caused by the intrinsically high nucleophilicity and redox sensitivity of thiol‐containing compounds, the physiological functions of many RLDs remain to be explored. Here, we focus on a single domain Acinetobacter baumannii RLD (Ab‐RLD) associated with a desulfurase encapsulin which is able to store substantial amounts of sulfur inside its protein shell. We determine the 1.6 Å x‐ray crystal structure of Ab‐RLD, highlighting a homodimeric structure with a number of unusual features. We show through kinetic analysis that Ab‐RLD exhibits thiosulfate sulfurtransferase activity with both cyanide and glutathione acceptors. Using native mass spectrometry and in vitro assays, we provide evidence that Ab‐RLD can stably carry a persulfide and thiosulfate modification and may employ a ternary catalytic mechanism. Our results will inform future studies aimed at investigating the functional link between Ab‐RLD and the desulfurase encapsulin.
Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the ...structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,β-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.
Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of ...synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
Compartmentalization is an essential feature of all cells. It allows cells to segregate and coordinate physiological functions in a controlled and ordered manner. Different mechanisms of ...compartmentalization exist, with the most relevant to prokaryotes being encapsulation via self‐assembling protein‐based compartments. One widespread example of such is that of encapsulins—cage‐like protein nanocompartments able to compartmentalize specific reactions, pathways, and processes in bacteria and archaea. While still relatively nascent bioengineering tools, encapsulins exhibit many promising characteristics, including a number of defined compartment sizes ranging from 24 to 42 nm, straightforward expression, the ability to self‐assemble via the Hong Kong 97‐like fold, marked physical robustness, and internal and external handles primed for rational genetic and molecular manipulation. Moreover, encapsulins allow for facile and specific encapsulation of native or heterologous cargo proteins via naturally or rationally fused targeting peptide sequences. Taken together, the attributes of encapsulins promise substantial customizability and broad usability. This review discusses recent advances in employing engineered encapsulins across various fields, from their use as bionanoreactors to targeted delivery systems and beyond. A special focus will be provided on the rational engineering of encapsulin systems and their potential promise as biomolecular research tools.
Encapsulins have recently been discovered to possess numerous attributes that make them highly customizable and valuable for various bioengineering and synthetic biology applications. Here, the authors review recent advances in the biology and rational engineering of encapsulins across various fields, including their use as bionanoreactors, reporters, targeted delivery systems, and platforms for nano‐ and biomaterials research. Further, possible future directions for encapsulin research as well as promising application directions are discussed.
Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress ...resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated by specific cargo protein motifs known as targeting peptides (TPs), though the structural basis for encapsulation of the largest encapsulin cargo class, dye-decolorizing peroxidases (DyPs), is currently unknown. Here, we characterize a DyP-containing encapsulin from the enterobacterial pathogen Klebsiella pneumoniae. By combining cryo-electron microscopy with TP and TP-binding site mutagenesis, we elucidate the molecular basis for cargo encapsulation. TP binding is mediated by cooperative hydrophobic and ionic interactions as well as shape complementarity. Our results expand the molecular understanding of enzyme encapsulation inside protein nanocompartments and lay the foundation for rationally modulating encapsulin cargo loading for biomedical and biotechnological applications.
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•Co-expression of a small non-native cargo results in high encapsulin cargo loading.•High cargo loading induces substantial heterogeneity of encapsulin shells.•The 2.53 Å cryo-EM ...structure of a highly cargo-loaded T3 encapsulin is presented.•The targeting peptide-shell interaction could be confidently modelled.
Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems’ structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.
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