Protein HU Research Articles

Application of virtual screening and molecular dynamics for the analysis of selectivity of inhibitors of HU proteins targeted to the DNA-recognition site

DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

Identification of nucleoid associated proteins (NAPs) under oxidative stress in Staphylococcus aureus

BackgroundBacterial nucleoid consists of genome DNA, RNA, and hundreds of nucleoid-associated proteins (NAPs). Escherichia coli nucleoid is compacted towards the stationary phase, replacing most log-phase NAPs with the major stationary-phase nucleoid protein, Dps. In contrast, Staphylococcus aureus nucleoid sustains the fiber structures throughout the growth. Instead, the Dps homologue, MrgA, expresses under oxidative stress conditions to clump the nucleoid, but the composition of the clumped nucleoid was elusive.ResultsThe staphylococcal nucleoid under oxidative stress was isolated by sucrose gradient centrifugation, and the proteins were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We identified 299 proteins in the nucleoid under oxidative stress, including 113 csNAPs (contaminant-subtracted NAPs). Comparison with the previously identified csNAPs in log- and stationary phase indicated that one fifth of the csNAPs under oxidative stress were the constitutive nucleoid components; importantly, several factors including HU, SarA, FabZ, and ribosomes were sustained under oxidative stress. Some factors (e.g. SA1663 and SA0092/SA0093) with unknown functions were included in the csNAPs list specifically under oxidative stress condition.ConclusionNucleoid constitutively holds Hu, SarA, FabG, and ribosomal proteins even under the oxidative stress, reflecting the active functions of the clumped nucleoid, unlikely to the dormant E. coli nucleoid compacted in the stationary phase or starvation.

CGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders.

Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP). Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro. Here we show that cGAS is activated in a strongly DNA length-dependent manner both in vitro and in human cells. We also show that cGAS dimers form ladder-like networks with DNA, leading to cooperative sensing of DNA length: assembly of the pioneering cGAS dimer between two DNA molecules is ineffective; but, once formed, it prearranges the flanking DNA to promote binding of subsequent cGAS dimers. Remarkably, bacterial and mitochondrial nucleoid proteins HU and mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 protein (HMGB1), can strongly stimulate long DNA sensing by cGAS. U-turns and bends in DNA induced by these proteins pre-structure DNA to nucleate cGAS dimers. Our results suggest a nucleation-cooperativity-based mechanism for sensitive detection of mitochondrial DNA and pathogen genomes, and identify HMGB/TFAM proteins as DNA-structuring host factors. They provide an explanation for the peculiar cGAS dimer structure and suggest that cGAS preferentially binds incomplete nucleoid-like structures or bent DNA.

Structure of DNA-binding HU protein from micoplasma Spiroplasma melliferum

Structure of DNA-binding HU protein from micoplasma Spiroplasma melliferum

Diarylethenes Display In Vitro Anti-TB Activity and Are Efficient Hits Targeting the Mycobacterium tuberculosis HU Protein.

Tuberculosis continues to be a great source of concern in global health because of the large reservoir of humans infected with the bacilli and the appearance of clinical isolates resistant to a wide array of anti-tuberculosis drugs. New drugs with novel mechanisms of action on new targets are urgently required to reduce global tuberculosis burden. Mycobacterium tuberculosis nucleoid associated protein (NAP) HU has been shown to be druggable and essential for the organism’s survival. In this study, four diarylethenes were synthesized using a one-pot decarboxylated Heck-coupling of coumaric acids with iodoanisoles. The prepared compounds 1–4 were tested for their in vitro growth inhibition of M. tuberculosis H37Rv using the spot culture growth inhibition assay, displaying minimum inhibitory concentrations between 9 and 22 µM. Their cytotoxicity against BHK-21 cell line showed half inhibition at concentrations between 98 and 729 µM. The most selective hit (SI = 81), demonstrated inhibition of M. tuberculosis HU protein involved in maintaining bacterial genome architecture.

Synchronous Adie's syndrome and type 1 antineuronal nuclear antibody (anti-Hu)-related paraneoplastic neurological syndromes as predictors of complete response in limited-stage small-cell lung cancer: A case report.

Adie's syndrome (AS) and paraneoplastic sensorimotor neuropathy with cerebellar ataxia (PSN CA) are extremely rare, rapidly progressive, autoimmune diseases associated with the development of antibodies against neuronal-specific Hu proteins that are abnormally expressed in small-cell lung cancer (SCLC). We herein present the unique case of a 55-year-old obese woman, previous heavy smoker, who, during treatment with standard cisplatin-etoposide chemotherapy for limited-stage SCLC, developed simultaneous AS and worsening symptoms consistent with PSN CA that led to significant neurological disability and severe axonal electrophysiological pattern on nerve conduction studies. Serology confirmed the presence of low-titre type 1 antineuronal nuclear antibodies (ANNA-1), previously referred to as anti-Hu antibodies. Following plasmapheresis, immunosuppressive therapy and physical rehabilitation, the neurological symptoms progressively improved. The tumour completely regressed, with no recurrence detected on subsequent radiological examinations. The aim of this case was to highlight the importance of a multidisciplinary team approach for early recognition and rapid treatment of paraneoplastic neurological syndromes (PNS) as key to achieving significant recovery and marked improvement of the neurological deficit. This report extends the literature by confirming earlier studies showing that the presence of serum ANNA-1 in SCLC, an aggressive type of pulmonary carcinoma that is challenging to treat, may portend a more favourable prognosis and response to chemotherapy. Thus, patients with SCLC and new-onset neurological symptoms should be tested for ANNA-1. The role of a multimodality approach to treating PNS is also emphasized.

Anchorless cell surface proteins function as laminin-binding adhesins in Lactobacillus rhamnosus FSMM22.

Anchorless cell surface proteins (CSPs) were extracted with 1 M lithium chloride solution from Lactobacillus rhamnosus FSMM22. Loss of the anchorless CSPs resulted in a 2-fold decrease in FSMM22 cells bound to a constitutive extracellular matrix glycoprotein, laminin, in vitro. DNA-binding protein HU, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and 30S ribosomal protein S19 (RpsS) were identified by mass spectrometry in the extract as laminin-binding adhesins. Among the four proteins, RpsS was immunohistochemically confirmed to exist on the cell surface. Our findings strongly suggest that anchorless CSPs can enhance bacterial adhesion to the host.

The histone-like protein HU has a role in gene expression during the acid adaptation response in Helicobacter pylori.

Gastritis, ulcers, and gastric malignancy have been linked to human gastric epithelial colonization by Helicobacter pylori. Characterization of the mechanisms by which H.pylori adapts to the human stomach environment is of crucial importance to understand H.pylori pathogenesis. In an effort to extend our knowledge of these mechanisms, we used proteomic analysis and qRT-PCR to characterize the role of the histone-like protein HU in the response of H.pylori to low pH. Proteomic analysis revealed that genes involved in chemotaxis, oxidative stress, or metabolism are under control of the HU protein. Also, expression of the virulence factors Ggt and NapA is affected by the null mutation of hup gene both at neutral and acid pH, as evidenced by qRT-PCR analysis. Those results showed that H.pylori gene expression is altered by shift to low pH, thus confirming that acid exposure leads to profound changes in genomic expression, and suggest that the HU protein is a regulator that may help the bacterium adapt to the acid stress. In accordance with previous reports, we found that the HU protein participates in gene expression regulation when the microorganism is exposed to acid stress. Such transcriptional regulation underlies protein accumulation in the H.pylori cell.

Enhanced conformational flexibility of the histone-like (HU) protein from Mycoplasma gallisepticum

The histone-like (HU) protein is one of the major nucleoid-associated proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This small positively charged dimeric protein binds DNA in a non-sequence specific manner promoting DNA super-structures. The majority of HU proteins are highly conserved among bacteria; however, HU protein from Mycoplasma gallisepticum (HUMgal) has multiple amino acid substitutions in the most conserved regions, which are believed to contribute to its specificity to DNA targets unusual for canonical HU proteins. In this work, we studied the structural dynamic properties of the HUMgal dimer by NMR spectroscopy and MD simulations. The obtained all-atom model displays compliance with the NMR data and confirms the heterogeneous backbone flexibility of HUMgal. We found that HUMgal, being folded into a dimeric conformation typical for HU proteins, has a labile α-helical body with protruded β-stranded arms forming DNA-binding domain that are highly flexible in the absence of DNA. The amino acid substitutions in conserved regions of the protein are likely to affect the conformational lability of the HUMgal dimer that can be responsible for complex functional behavior of HUMgal in vivo, e.g. facilitating its spatial adaptation to non-canonical DNA-targets.

Structural and evolutionary analyses reveal determinants of DNA binding specificities of nucleoid-associated proteins HU and IHF

Nucleoid-associated proteins (NAPs) are chromosome-organizing factors, which affect the transcriptional landscape of a bacterial cell. HU is an NAP, which binds to DNA with a broad specificity while homologous IHF (Integration Host Factor), binds DNA with moderately higher specificity. Specificity and differential binding affinity of HU/IHF proteins towards their target binding sites play a crucial role in their regulatory dynamics. Decades of biochemical and genomic studies have been carried out for HU and IHF like proteins. Yet, questions related to their DNA binding specificity, and differential ability to bend DNA thus affecting the binding site length remained unanswered. In addition, the problem has not been investigated from an evolutionary perspective. Our phylogenetic analysis revealed three major clades belonging to HU, IHFα and IHFβ like proteins with reference to E. coli. We carried out a comparative analysis of three-dimensional structures of HU/IHF proteins to gain insight into the structural basis of clade division. The present study revealed three major features which contribute to differential DNA binding specificity of HU/IHF proteins, (I) conformational restriction of DNA binding residues due to salt-bridge formation, (II) the enrichment of alanine in the DNA binding site increasing conformational space of flexible side chains in its vicinity and (III) nature of DNA binding residue (Arg to Lys bias in different clades) which interacts differentially to DNA bases. We observed an extended electropositive surface at the DNA draping site for IHF clade proteins compared to HU, which stabilizes the DNA bend. Differences in the dimer stabilization strategies between HU and IHF were also observed. Our analysis reveals a comprehensive evolutionary picture, which rationalizes the origin of multi-specificity of HU/IHF proteins using sequence and structure-based determinants, which could also be applied to understand differences in binding specificities of other nucleic acid binding proteins.

Structural basis of the high thermal stability of the histone-like HU protein from the mollicute Spiroplasma melliferum KC3.

The three-dimensional structure of the histone-like HU protein from the mycoplasma Spiroplasma melliferum KC3 (HUSpm) was determined at 1.4 Å resolution, and the thermal stability of the protein was evaluated by differential scanning calorimetry. A detailed analysis revealed that the three-dimensional structure of the HUSpm dimer is similar to that of its bacterial homologues but is characterized by stronger hydrophobic interactions at the dimer interface. This HUSpm dimer interface lacks salt bridges but is stabilized by a larger number of hydrogen bonds. According to the DSC data, HUSpm has a high denaturation temperature, comparable to that of HU proteins from thermophilic bacteria. To elucidate the structural basis of HUSpm thermal stability, we identified amino acid residues potentially responsible for this property and modified them by site-directed mutagenesis. A comparative analysis of the melting curves of mutant and wild-type HUSpm revealed the motifs that play a key role in protein thermal stability: non-conserved phenylalanine residues in the hydrophobic core, an additional hydrophobic loop at the N-terminal region of the protein, the absence of the internal cavity present at the dimer interface of some HU proteins, and the presence of additional hydrogen bonds between the monomers that are missing in homologous proteins.

Ribosome profiling reveals an adaptation strategy of reduced bacterium to acute stress

Bacteria of class Mollicutes (mycoplasmas) feature significant genome reduction which makes them good model organisms for systems biology studies. Previously we demonstrated, that drastic transcriptional response of mycoplasmas to stress results in a very limited response on the level of protein. In this study we used heat stress model of M. gallisepticum and ribosome profiling to elucidate the process of genetic information transfer under stress. We found that under heat stress ribosomes demonstrate selectivity towards mRNA binding. We identified that heat stress response may be divided into two groups on the basis of absolute transcript abundance and fold-change in the translatome. One represents a noise-like response and another is likely an adaptive one. The latter include ClpB chaperone, cell division cluster, homologs of immunoblocking proteins and short ORFs with unknown function. We found that previously identified read-through of terminators contributes to the upregulation of transcripts in the translatome as well. In addition we identified that ribosomes of M. gallisepticum undergo reorganization under the heat stress. The most notable event is decrease of the amount of associated HU protein. In conclusion, only changes of few adaptive transcripts significantly impact translatome, while widespread noise-like transcription plays insignificant role in translation during stress.

A specific single-stranded DNA induces a distinct conformational change in the nucleoid-associated protein HU

In prokaryotic cells, genomic DNA forms an aggregated structure with various nucleoid-associated proteins (NAPs). The functions of genomic DNA are cooperatively modulated by NAPs, of which HU is considered to be one of the most important. HU binds double-stranded DNA (dsDNA) and serves as a structural modulator in the genome architecture. It plays important roles in diverse DNA functions, including replication, segregation, transcription and repair. Interestingly, it has been reported that HU also binds single-stranded DNA (ssDNA) regardless of sequence. However, structural analysis of HU with ssDNA has been lacking, and the functional relevance of this binding remains elusive.In this study, we found that ssDNA induced a significant change in the secondary structure of Thermus thermophilus HU (TtHU), as observed by analysis of circular dichroism spectra. Notably, this change in secondary structure was sequence specific, because the complementary ssDNA or dsDNA did not induce the change. Structural analysis using nuclear magnetic resonance confirmed that TtHU and this ssDNA formed a unique structure, which was different from the previously reported structure of HU in complex with dsDNA. Our data suggest that TtHU undergoes a distinct structural change when it associates with ssDNA of a specific sequence and subsequently exerts a yet-to-be-defined function.

Genes on a Wire: The Nucleoid-Associated Protein HU Insulates Transcription Units in Escherichia coli.

The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling.

Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding.

HU histone-like DNA-binding protein from Thermus thermophilus: structural and evolutionary analyses.

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus-Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.

Genome scale patterns of supercoiling in a bacterial chromosome.

DNA in bacterial cells primarily exists in a negatively supercoiled state. The extent of supercoiling differs between regions of the chromosome, changes in response to external conditions and regulates gene expression. Here we report the use of trimethylpsoralen intercalation to map the extent of supercoiling across the Escherichia coli chromosome during exponential and stationary growth phases. We find that stationary phase E. coli cells display a gradient of negative supercoiling, with the terminus being more negatively supercoiled than the origin of replication, and that such a gradient is absent in exponentially growing cells. This stationary phase pattern is correlated with the binding of the nucleoid-associated protein HU, and we show that it is lost in an HU deletion strain. We suggest that HU establishes higher supercoiling near the terminus of the chromosome during stationary phase, whereas during exponential growth DNA gyrase and/or transcription equalizes supercoiling across the chromosome.

Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization.

Nucleoid-associated protein HU, a conserved protein across eubacteria is necessary for maintaining the nucleoid organization and global regulation of gene expression. Mycobacterium tuberculosis HU (MtHU) is distinct from the other orthologues having 114 amino acid long carboxyl terminal extensions with a high degree of sequence similarity to eukaryotic histones. In this study, we demonstrate that the DNA binding property of MtHU is regulated by posttranslational modifications akin to eukaryotic histones. MtHU purified from M. tuberculosis cells is found to be acetylated on multiple lysine residues unlike the E. coli expressed recombinant protein. Using coimmunoprecipitation assay, we identified Eis as one of the acetyl transferases that interacts with MtHU and modifies it. Although Eis is known to acetylate aminoglycosides, the kinetics of acetylation showed that its protein acetylation activity on MtHU is robust. In vitro Eis modified MtHU at various lysine residues, primarily those located at the carboxyl terminal domain. Acetylation of MtHU caused reduced DNA interaction and alteration in DNA compaction ability of the NAP. Over-expression of the Eis leads to hyperacetylation of HU and decompaction of genome. These results provide first insights into the modulation of the nucleoid structure by lysine acetylation in bacteria.

Electrophysiological and morphological changes in colonic myenteric neurons from chemotherapy-treated patients: a pilot study.

BackgroundPatients receiving anticancer chemotherapy experience a multitude of gastrointestinal side‐effects. However, the causes of these symptoms are uncertain and whether these therapeutics directly affect the enteric nervous system is unknown. Our aim was to determine whether the function and morphology of myenteric neurons are altered in specimens of the colon from chemotherapy‐treated patients.MethodsColon specimens were compared from chemotherapy‐treated and non‐treated patients following colorectal resections for removal of carcinoma. Intracellular electrophysiological recordings from myenteric neurons and immunohistochemistry were performed in whole mount preparations.Key ResultsMyenteric S neurons from chemotherapy‐treated patients were hyperexcitable; more action potentials (11.4 ± 9.4, p < 0.05) were fired in response to depolarising current pulses than in non‐treated patients (1.4 ± 0.5). The rheobase and the threshold to evoke action potentials were significantly lower for neurons from chemotherapy‐treated patients compared to neurons from non‐treated patients (p < 0.01). Fast excitatory postsynaptic potential reversal potential was more positive in neurons from chemotherapy‐treated patients (p < 0.05). An increase in the number of neurons with translocation of Hu protein from the cytoplasm to the nucleus was observed in specimens from chemotherapy‐treated patients (103 ± 25 neurons/mm2, 37.2 ± 7.0%, n = 8) compared to non‐treated (26 ± 5 neurons/mm2, 11.9 ± 2.7%, n = 12, p < 0.01). An increase in the soma size of neuronal nitric oxide synthase‐immunoreactive neurons was also observed in these specimens.Conclusions & InferencesThis is the first study suggesting functional and structural changes in human myenteric neurons in specimens of colon from patients receiving anticancer chemotherapy. These changes may contribute to the causation of gastrointestinal symptoms experienced by chemotherapy‐treated patients.

CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications.

For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.