C(P)XCG Proteins of Haloferax volcanii with Predicted Zinc Finger Domains: The Majority Bind Zinc, but Several Do Not
Ontology highlight
ABSTRACT:
SUBMITTER: Uresin D
PROVIDER: S-EPMC11241148 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Zinc finger domains are highly structured and can mediate interactions to DNA, RNA, proteins, lipids, and small molecules. Accordingly, zinc finger proteins are very versatile and involved in many biological functions. Eukaryotes contain a wealth of zinc finger proteins, but zinc finger proteins have also been found in archaea and bacteria. Large zinc finger proteins have been well studied, however, in stark contrast, single domain zinc finger µ-proteins of less than 70 amino acids have not been studied at all, with one single exception. Therefore, 16 zinc finger µ-proteins of the haloarchaeon Haloferax volcanii were chosen and in frame deletion mutants of the cognate genes were generated. The phenotypes of mutants and wild-type were compared under eight different conditions, which were chosen to represent various pathways and involve many genes. None of the mutants differed from the wild-type under optimal or near-optimal conditions. However, 12 of the 16 mutants exhibited a phenotypic difference under at least one of the four following conditions: Growth in synthetic medium with glycerol, growth in the presence of bile acids, biofilm formation, and swarming. In total, 16 loss of function and 11 gain of function phenotypes were observed. Five mutants indicated counter-regulation of a sessile versus a motile life style in H. volcanii. In conclusion, the generation and analysis of a set of deletion mutants demonstrated the high importance of zinc finger µ-proteins for various biological functions, and it will be the basis for future mechanistic insight.
| S-EPMC6562870 | biostudies-literature
Project description:It is increasingly recognized that very small proteins (μ-proteins) are ubiquitously found in all species of the three domains of life, and that they fulfill important functions. The halophilic archaeon Haloferax volcanii contains 282 μ-proteins of less than 70 amino acids. Notably, 43 of these contain two C(P)XCG motifs, suggesting their potential to complex a zinc ion. To explore the significance of these proteins, 16 genes encoding C(P)XCG proteins had been deleted, and the majority of mutants exhibited phenotypic differences to the wild-type. One such protein, HVO_2753, was thoroughly characterized in a previous study. In the present study an in-depth analysis of a second protein, HVO_0758, was performed. To achieve this goal, the HVO_0758 protein was produced heterologously in Escherichia coli and homologously in H. volcanii. The purified protein was characterized using various biochemical approaches and NMR spectroscopy. The findings demonstrated that HVO_0758 is indeed a bona fide zinc finger protein, and that all four cysteine residues are essential for folding. The NMR solution structure was solved, revealing that HVO_0758 is comprised of an N-terminal alpha helix containing several positively charged residues and a globular core with the zinc finger domain. The transcriptomes of the HVO_0758 deletion mutant and, for comparison, the HVO_2753 deletion mutant were analyzed with RNA-Seq and compared against that of the wild-type. In both mutants many motility and chemotaxis genes were down-regulated, in agreement to the phenotype of the deletion mutants, which had a swarming deficit. The two H. volcanii zinc-finger μ-proteins HVO_0758 and HVO_2753 showed many differences. Taken together, two zinc finger μ-proteins of H. volcanii have been characterized intensively, which emerged as pivotal contributors to swarming behavior and biofilm formation.
| S-EPMC10716476 | biostudies-literature
Project description:Transcriptome analysis based on total RNA-seq was performed on different Haloferax volcanii strains including mutants strains iincluding deletion strains for two small proteins. Differential expression analysis showed that a subset of genes were found to be regulated in the absence of the small proteins.
2023-11-13 | GSE228855 | GEO
Project description:Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a beta-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the epsilon-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.
| S-EPMC2872088 | biostudies-literature
Project description:Proteasomes are composed of 20S core particles (CPs) of alpha- and beta-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of alpha1 and alpha2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including alpha1 Thr147, alpha2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to alpha1, thus, revealing a new type of proteasomal modification. Probing the biological role of alpha1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for alpha1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to alpha1. The alpha1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.
| S-EPMC2910475 | biostudies-literature
Project description:20S proteasomes are large, multicatalytic proteases that play an important role in intracellular protein degradation. The barrel-like architecture of 20S proteasomes, formed by the stacking of four heptameric protein rings, is highly conserved from archaea to eukaryotes. The outer two rings are composed of alpha-type subunits, and the inner two rings are composed of beta-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different alpha-type proteins, alpha1 and alpha2, and one beta-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins (alpha1, alpha2, and beta) are modified either post- or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the beta subunit was identified at Ser129 of the deduced protein sequence. In addition, alpha1 and alpha2 contained N-terminal acetyl groups. These findings represent the first evidence of acetylation and phosphorylation of archaeal proteasomes and are one of the limited examples of post- and/or cotranslational modification of proteins in this unusual group of organisms.
| S-EPMC1636277 | biostudies-literature
Project description:A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3). This cytochrome aa(3) consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.
| S-EPMC139504 | biostudies-literature
Project description:Extracellular DNA is found in all environments and is a dynamic component of the microbial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA concentrations measured in nature-a potential rich source of carbon, nitrogen, and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration, and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent, and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA. Additionally, fluorescence microscopy showed that labeled DNA co-localized with H. volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in DNA processing at the cell surface, and deletion of Hvo_1477 created a strain deficient in the ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.
| S-EPMC3929857 | biostudies-literature
Project description:Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.
| S-EPMC2676566 | biostudies-literature
Project description:To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated). Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I-III) and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA) maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.
| S-EPMC4390866 | biostudies-literature