Prokaryotic Proteins Research Articles

Small Prokaryotic DNA-Binding Proteins Protect Genome Integrity throughout the Life Cycle.

Genomes of all organisms are persistently threatened by endogenous and exogenous assaults. Bacterial mechanisms of genome maintenance must provide protection throughout the physiologically distinct phases of the life cycle. Spore-forming bacteria must also maintain genome integrity within the dormant endospore. The nucleoid-associated proteins (NAPs) influence nucleoid organization and may alter DNA topology to protect DNA or to alter gene expression patterns. NAPs are characteristically multifunctional; nevertheless, Dps, HU and CbpA are most strongly associated with DNA protection. Archaea display great variety in genome organization and many inhabit extreme environments. As of yet, only MC1, an archaeal NAP, has been shown to protect DNA against thermal denaturation and radiolysis. ssDNA are intermediates in vital cellular processes, such as DNA replication and recombination. Single-stranded binding proteins (SSBs) prevent the formation of secondary structures but also protect the hypersensitive ssDNA against chemical and nuclease degradation. Ionizing radiation upregulates SSBs in the extremophile Deinococcus radiodurans.

Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA

SummaryArgonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

Characterization of nanog in Nile tilapia (Oreochromis niloticus) and its spatiotemporal expression patterns during embryonic and gonadal development.

Nanog is one of the well-characterized core transcription factors in pluripotency maintenance network. So far, studies on fishes indicate that the Nanog expression occurs from embryonic 1-cell stage to blastula stage, and is restricted to the gonadal germline cells in adult tissues, which is strikingly different from that in mammals. However, whether this expression profile is conservative in fishes remains to be investigated. Here Nile tilapia (Oreochromis niloticus) nanog (named as Ong) was identified and its spatiotemporal expression patterns during embryonic and gonadal development were investigated. The Ong cDNA contains an open reading frame of 678bp, encoding 226 amino acids. The anti-Ong antibody was prepared through prokaryotic protein expression and its specificity was validated. The Ong expression in embryonic 1-cell stage did not appear until the early stage of blastocyst and continued to the late stage of blastocyst. In adult tissues, its expression was limited to gonads. Its expression patterns during gonadal development were further investigated by in situ hybridization and immunohistochemical staining. In testis, Ong was not expressed at 30 dah (days after hatching), but highly expressed in spermatogonia and spermatocytes at 150 dah; in ovaries at 30 and 150 dah, it was not expressed in germline cells but in all somatic cells. This expression profile is strikingly different from reports in fishes to date. Our study firstly indicates that the Nanog expression profile is not conservative in fishes. This study is valuable for further functional and evolutionary study of this gene.

Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo.

ABSTRACTThe Argonaute proteins are present in all three domains of life, which are archaea, bacteria, and eukarya. Unlike the eukaryotic Argonaute proteins, which use small RNA guides to target mRNAs, some prokaryotic Argonaute proteins (pAgos) use a small DNA guide to interfere with DNA and/or RNA targets. However, the mechanisms of pAgo natural function remain unknown. Here, we investigate the mechanism by which pAgo from Natronobacterium gregoryi (NgAgo) targets plasmid and bacteriophage T7 DNA using a heterologous Escherichia coli-based model system. We show that NgAgo expressed from a plasmid linearizes its expression vector. Cotransformation assays demonstrate that NgAgo requires an RNA in trans that is transcribed from the bacteriophage T7 promoter to activate cleavage of a cotransformed plasmid, reminiscent of the trans-RNA function in CRISPR/Cas9. We propose a mechanism to explain how NgAgo eliminates invading foreign DNA and bacteriophage. By leveraging this discovery, we show that NgAgo can be programmed to target a plasmid or a chromosome locus.

Prokaryotic Collagen-Like Proteins as Novel Biomaterials.

Collagens are the major structural component in animal extracellular matrices and are critical signaling molecules in various cell-matrix interactions. Its unique triple helical structure is enabled by tripeptide Gly-X-Y repeats. Understanding of sequence requirements for animal-derived collagen led to the discovery of prokaryotic collagen-like protein in the early 2000s. These prokaryotic collagen-like proteins are structurally similar to mammalian collagens in many ways. However, unlike the challenges associated with recombinant expression of mammalian collagens, these prokaryotic collagen-like proteins can be readily expressed in E. coli and are amenable to genetic modification. In this review article, we will first discuss the properties of mammalian collagen and provide a comparative analysis of mammalian collagen and prokaryotic collagen-like proteins. We will then review the use of prokaryotic collagen-like proteins to both study the biology of conventional collagen and develop a new biomaterial platform. Finally, we will describe the application of Scl2 protein, a streptococcal collagen-like protein, in thromboresistant coating for cardiovascular devices, scaffolds for bone regeneration, chronic wound dressing and matrices for cartilage regeneration.

Structural basis for the catalytic activity of filamentous human serine beta-lactamase-like protein LACTB

Serine beta-lactamase-like protein (LACTB) is a mammalian mitochondrial serine protease that can specifically hydrolyze peptide bonds adjacent to aspartic acid residues and is structurally related to prokaryotic penicillin-binding proteins. Here, we determined the cryoelectron microscopy structures of human LACTB (hLACTB) filaments from wild-type protein, a middle region deletion mutant, and in complex with the inhibitor Z-AAD-CMK at 3.0-, 3.1-, and 2.8-Å resolution, respectively. Structural analysis and activity assays revealed that three interfaces are required for the assembly of hLACTB filaments and that the formation of higher order helical structures facilitates its cleavage activity. Further structural and enzymatic analyses of middle region deletion constructs indicated that, while this region is necessary for substrate hydrolysis, it is not required for filament formation. Moreover, the inhibitor-bound structure showed that hLACTB may cleave peptide bonds adjacent to aspartic acid residues. These findings provide the structural basis underlying hLACTB catalytic activity.

Sumoylation in Physiology, Pathology and Therapy.

Sumoylation is an essential post-translational modification that has evolved to regulate intricate networks within emerging complexities of eukaryotic cells. Thousands of target substrates are modified by SUMO peptides, leading to changes in protein function, stability or localization, often by modulating interactions. At the cellular level, sumoylation functions as a key regulator of transcription, nuclear integrity, proliferation, senescence, lineage commitment and stemness. A growing number of prokaryotic and viral proteins are also emerging as prime sumoylation targets, highlighting the role of this modification during infection and in immune processes. Sumoylation also oversees epigenetic processes. Accordingly, at the physiological level, it acts as a crucial regulator of development. Yet, perhaps the most prominent function of sumoylation, from mammals to plants, is its role in orchestrating organismal responses to environmental stresses ranging from hypoxia to nutrient stress. Consequently, a growing list of pathological conditions, including cancer and neurodegeneration, have now been unambiguously associated with either aberrant sumoylation of specific proteins and/or dysregulated global cellular sumoylation. Therapeutic enforcement of sumoylation can also accomplish remarkable clinical responses in various diseases, notably acute promyelocytic leukemia (APL). In this review, we will discuss how this modification is emerging as a novel drug target, highlighting from the perspective of translational medicine, its potential and limitations.

Alg mannosyltransferases: From functional and structural analyses to the lipid-linked oligosaccharide pathway reconstitution

BackgroundN-glycosylation is initiated from the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER), which is catalyzed by a series of Alg (asparagine-linked glycosylation) proteins. Scope of reviewThis review summarizes our recent studies on the enzymology of Alg mannosyltransferases (MTases). We also discuss the membrane topology and physiological importance of several ER cytosolic Alg proteins. Major conclusionsUtilizing an efficient prokaryotic protein expression system and a new LC–MS quantitative activity assay, we overexpressed all Alg MTases and performed enzymology studies. Moreover, by reconstituting the LLO pathway, the high-yield chemoenzymatic synthesis of high-mannose-type N-glycans was accomplished using recombinant Alg MTases. General significanceThe analysis of the enzymology and topology of Alg MTases has provided valuable biochemical information in the LLO biosynthesis pathway. In addition, an efficient chemoenzymatic strategy that could prepare various oligomannose-type N-glycans in sufficient amounts was established for further biological assays.

Backbone NMR resonance assignment of the intrinsically disordered UBact protein from Nitrospira nitrosa.

Ubiquitin signaling in eukaryotes is responsible for a variety of cellular outcomes, most notably proteasomal degradation. A recent bioinformatic study has revealed the existence of a new proteasomal operon in certain gram-negative bacteria phyla. This operon contains genes similar to those included in the prokaryotic ubiquitin-like protein (Pup) proteasomal operon, but do not themselves contain Pup. Instead, they encode for a protein termed UBact with 30% sequence similarity to Pup. Here, we report the near-complete NMR assignment of the backbone and partial assignment of the side chain chemical shifts of the UBact protein from Nitrospira nitrosa. The 1H-15N HSQC spectrum shows a narrow spread of proton NMR signals, characteristic of an intrinsically disordered protein. This chemical shift assignment will facilitate further NMR studies to explore the role of UBact in this new putative proteasomal operon.

Prokaryotic cytoskeletal filaments: a tool to build a minimal DNA segregation system for synthetic cells

Microtubules are eukaryotic cytoskeletal filaments that play a crucial role in cell division and intracellular transportation. In recent years, cytoskeletal filaments which share a number of characteristic features with eukaryotic microtubules have been discovered in prokaryotic cells. From cell division to DNA segregation, prokaryotic tubulin-like proteins participate in various functions by forming filamentous structures. A synthetic cell is an artificial lipid bilayer container which encloses a cell free gene-expression system that has at least two main traits of a living cell: the ability of autonomous growth and division.

Evolutionary History of RNA Modifications at N6-Adenosine Originating from the R-M System in Eukaryotes and Prokaryotes.

Simple SummaryThe m6A is the most abundant and well-studied modification of mRNA, and plays an important role in transcription and translation. It is known to be evolutionarily conserved machinery present in the last eukaryotic common ancestor (LECA). The writers and erasers responsible for adding or removing m6A belong to specific protein families, respectively, suggesting that these members are evolutionarily related. However, only some of these mRNA m6A modification-associated proteins have been studied from an evolutionary perspective, while there has been no comprehensive and systematic analysis of the distributions and evolutionary history of N6mA-associated proteins in the three kingdoms of life. In this study, we identified orthologues of all the reported N6mA-associated proteins in 88 organisms from three kingdoms of life and comprehensively reconstructed the evolutionary history of the RNA N6mA modification machinery. The results demonstrate that RNA N6mA-MTases are derived from at least two different types of prokaryotic DNA MTases (class α and β MTases). As the m6A reader, YTH proteins may be acquired by LECA from an individual prokaryotic YTH-domain protein that evolved from the N-terminals of an R-M system endonuclease. In addition, the origin of eukaryotic ALKBH family proteins is inferred to be driven by at least two occasions of independent HTG from the bacterial ALKB family.Methylation at the N6-position of adenosine (N6mA) on mRNA (m6A) is one of the most widespread, highly selective and dynamically regulated RNA modifications and plays an important role in transcription and translation. In the present study, a comprehensive analysis of phylogenetic relationships, conserved domain sequence characteristics and protein structure comparisons were employed to explore the distribution of RNA N6mA modification (m6A, m6,6A, m6Am, m6, 6Am and m6t6A)-associated proteins (writers, readers and erasers) in three kingdoms of life and reveal the evolutionary history of these modifications. These findings further confirmed that the restriction-modification (R-M) system is the origin of DNA and RNA N6mA modifications. Among them, the existing mRNA m6A modification system derived from the last eukaryotic common ancestor (LECA) is the evolutionary product of elements from the last universal common ancestor (LUCA) or driven by horizontal gene transfer (HGT) from bacterial elements. The subsequent massive gene gains and losses contribute to the development of unique and diverse functions in distinct species. Particularly, RNA methyltransferases (MTases) as the writer responsible for adding N6mA marks on mRNA and ncRNAs may have evolved from class α and β prokaryotic “orphan” MTases originating from the R-M system. The reader, YTH proteins that specifically recognize the m6A deposit, may be acquired by LECA from an individual prokaryotic YTH-domain protein that evolved from N-terminals of an R-M system endonuclease. The eraser, which emerged from the ALKB family (ALKBH5 and FTO) in eukaryotes, may be driven by independent HTG from bacterial ALKB proteins. The evolutionary history of RNA N6mA modifications was inferred in the present study, which will deepen our understanding of these modifications in different species.

Phenylalanine stacking enhances the red fluorescence of biliverdin IXα on UV excitation in sandercyanin fluorescent protein.

Biliverdin IXα (BV) binds to several prokaryotic and eukaryotic proteins. How nature exploits the versatility of BV's properties is not fully understood. Unlike free BV, the Sandercyanin fluorescent protein bound to BV (SFP-BV) shows enhanced red fluorescence (675 nm) on excitation in the UV region (380 nm). Site-directed mutagenesis showed that the BV complex of two SFP variants, F55A and E79A, resulted in the loss of red fluorescence. Crystal structures of the complexes of these proteins with BV show the absence of stacking interactions of the F55 phenyl ring with BV. BV changes from ZZZssa conformation in the wild-type to ZZZsss conformation in the variants. In the nonfluorescent mutants, the lowest excited state is destabilized, resulting in nonradiative decay.

Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.

Prokaryotic expression and solubilisation of Arabidopsis ROOT UVB SENSITIVE 1 from inclusion bodies in Escherichia coli

The RUS (ROOT UVB SENSITIVE 1) proteins characterized by their unique DUF647 domain are widely distributed in eukaryotes. Their functional roles are largely unknown except for the possible involvement of Arabidopsis RUS1 and RUS2 in early seedling development. To investigate the biochemical roles of the RUS proteins, full length and truncated Arabidopsis RUS1 were seamlessly fused with GFP and cloned into prokaryotic expression vector pQE-100 which allows proteins expressed with an N-terminal 6?His tag. Expression of the full length RUS1-GFP could not be detected after adding the inducer IPTG, while a truncated RUS1-GFP was expressed at high levels and formed inclusion bodies in Escherichia coli. The inclusion bodies were dissolved in a denaturing buffer, and then the truncated RUS1-GFP fusion protein in the supernatant was bound to a Ni-NTA slurry. The bound proteins were eluted after the non-specific binding proteins were washed away. The purified truncated proteins were detected as a single clear band of the expected size in SDS-PAGE, and were further confirmed by the Western blot test. Our results suggest that the impossible expression of the full length RUS1 protein in E. coli can be expressed in truncated form, and inclusion bodies can be effectively solubilized.

Production of Membrane Proteins in Pseudomonas stutzeri.

Functional and structural studies on membrane proteins are often hampered by insufficient yields, misfolding and aggregation during the production and purification process. Escherichia coli is the most commonly used expression host for the production of recombinant prokaryotic integral membrane proteins. However, in many cases expression hosts other than E. coli are more appropriate for certain target proteins. Here, we report a convenient, systematically developed expression system using the γ-proteobacterium Pseudomonas stutzeri as an alternative production host for over-expression of integral membrane proteins. P. stutzeri can be easily and inexpensively cultured in large quantities. The Pseudomonas expression vectors are designed for inducible expression of affinity-tagged fusion proteins controlled by the PBAD promoter. This chapter provides detailed protocols of the different steps required to successfully produce and isolate recombinant membrane proteins with high yields in P. stutzeri.

Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

The curious case of SARM1: Dr. Jekyll and Mr. Hyde in cell death and immunity?

Sterile alpha and toll/interleukin-1 receptor motif-containing protein 1 (SARM1) was first identified as a novel ortholog of Drosophila protein CG7915 and was subsequently placed as the fifth member of the human TIR-containing adaptor protein. SARM1 holds a unique position in this family where, unlike other members, it downregulates NFκB activity in response to immunogenic stimulation, interacts with another member of the family, TRIF, to negatively regulate its function, and it also mediates cell death responses. Over the past decade, SARM1 has emerged as one of the primary mediators of programmed axonal degeneration and this robust regulation of axonal degeneration-especially in models of peripheral neuropathy and traumatic injury-makes it an attractive target for therapeutic intervention. The TIR domain of SARM1 possesses an intrinsic NADase activity resulting in cellular energy deficits within the axons, a striking deviation from its other family members of human TLR adaptors. Interestingly, the TIR NADase activity, as seen in SARM1, is also observed in several prokaryotic TIR-containing proteins where they are involved in immune evasion once within the host. Although the immune function of SARM1 is yet to be conclusively discerned, this closeness in function with the prokaryotic TIR-domain containing proteins, places it at an interesting juncture of evolution raising questions about its origin and function in cell death and immunity. In this review, we discuss how a conserved immune adaptor protein like SARM1 switches to a pro-neurodegenerative function and the evolutionarily significance of the process.

A Nosy Neighbor: Purification and Functional Characterization of Lpg2149

Ubiquitination is a process that marks proteins for various cell-signaling pathways, namely protein degradation and other processes. Th ese pathways are essential in a wide array of cellular processes, including defense mechanisms against invading pathogens. Th e ubiquitination process is universally found in all eukaryotic organisms, including plants and animals, and thus plays a vital role in cellular homeostasis. Recently, more discoveries have been made on prokaryotic effector proteins that hijack the ubiquitination system even when they do not possess a ubiquitin system of their own. MavC, also known as lpg2147 (Gan, Nakayasu, Hollenbeck, & Luo, 2019; Puvar et al., 2020; Valleau et al., 2018), has been found to be a ubiquitinating enzyme that ubiquitinates UbE2N by bypassing the usual E1, E2, E3 ubiquitination pathway. MavC plays an important role in the infection of Legionella pneumophila, which is the culprit of Legionnaires’ disease. Th rough earlier molecular biology analysis, it has been discovered that a neighboring gene on the same locus as MavC encodes lpg2149, which has been characterized to inhibit MavC’s function. Given the novelty of this protein, this research project aims to achieve cloning, expression, and purification of lpg2149 so that in its inhibitory complex lpg2149 can be captured by X-ray crystallography. An attempt was made to crystallize and obtain the structure of lpg2149 with MavC. With the optimization of lpg2149 production demonstrated in this project, a better understanding of L. pneumophila pathogenesis can be obtained, which would help in our understanding of regulating L. pneumophila infection.

Identification of RNA Binding Partners of CRISPR-Cas Proteins in Prokaryotes Using RIP-Seq.

CRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome. Here, we describe RIP-seq as a method to globally identify the direct RNA binding partners of CRISPR-Cas RNPs using the Cas9 nuclease as an example. RIP-seq combines co-immunoprecipitation (coIP) of an epitope-tagged Cas9 followed by isolation and deep sequencing analysis of its co-purified bound RNAs. This method can not only be used to study interactions of Cas9 with its known interaction partners, crRNAs and tracrRNA in native systems, but also to reveal potential additional RNA substrates of Cas9. For example, in RIP-seq analysis of Cas9 from the foodborne pathogen Campylobacter jejuni (CjeCas9), we recently identified several endogenous RNAs bound to CjeCas9 RNP in a crRNA-dependent manner, leading to the discovery of PAM-independent RNA cleavage activity of CjeCas9as well as non-canonical crRNAs. RIP-seq can be easily adapted to any other effector RNP of choice from other CRISPR-Cas systems, allowing for the identification of target RNAs. Deciphering novel RNA-protein interactions for CRISPR-Cas proteins within host bacterial genomes will lead to a better understanding of the molecular mechanisms and functions of these systems and enable us to use the in vivo identified interaction rules as design principles for nucleic acid-targeting applications, fitted to each nuclease of interest.

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis.

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.