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Phosphorylation of Ser8 promotes zinc-induced dimerization of the amyloid-beta metal-binding domain

Kulikova, Alexandra A; Tsvetkov, Philipp O; Indeykina, Maria I; Popov, Igor A; Zhokhov, Sergey S; Golovin, Andrey V; Polshakov, Vladimir I; Kozin, Sergey A; Nudler, Evgeny; Makarov, Alexander A
Zinc-induced aggregation of the amyloid-beta peptide (Abeta) is a hallmark molecular feature of Alzheimer's disease (AD). Recently it was shown that phosphorylation of Abeta at Ser8 promotes the formation of toxic aggregates. In this work, we have studied the impact of Ser8 phosphorylation on the mode of zinc interaction with the Abeta metal-binding domain 1-16 using isothermal titration calorimetry, electrospray ionization mass spectrometry and NMR spectroscopy. We have discovered a novel zinc binding site ((6)HDpS(8)) in the phosphorylated peptide, in which the zinc ion is coordinated by the imidazole ring of His6, the phosphate group attached to Ser8 and a backbone carbonyl group of His6 or Asp7. Interaction of the zinc ion with this site involves His6, thereby withdrawing it from the interaction pattern observed in the non-modified peptide. This event was found to stimulate dimerization of peptide chains through the (11)EVHH(14) site, where the zinc ion is coordinated by the two pairs of Glu11 and His14 in the two peptide subunits. The proposed molecular mechanism of zinc-induced dimerization could contribute to the understanding of initiation of pathological Abeta aggregation, and the (11)EVHH(14) tetrapeptide can be considered as a promising drug target for the prevention of amyloidogenesis.
PMID: 25023775
ISSN: 1742-2051
CID: 1268492

Riboswitches in regulation of Rho-dependent transcription termination

Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny
Riboswitches are RNA sensors of small metabolites and ions that regulate gene expression in response to environmental changes. In bacteria, the riboswitch sensor domain usually controls the formation of a strong RNA hairpin that either functions as a potent transcription terminator or sequesters a ribosome-binding site. A recent study demonstrated a novel mechanism by which a riboswitch controls Rho-dependent transcription termination. This riboswitch mechanism is likely a widespread mode of gene regulation that determines whether a protein effector is able to attenuate transcription. This article is part of a Special Issue entitled: Riboswitches.
PMID: 24731855
ISSN: 0006-3002
CID: 1362732

Bacterial gasotransmitters: an innate defense against antibiotics

Luhachack, Lyly; Nudler, Evgeny
In recent decades, there has been growing interest in the field of gasotransmitters, endogenous gaseous signaling molecules (NO, H2S, and CO), as regulators of a multitude of biochemical pathways and physiological processes. Most of the concerted effort has been on eukaryotic gasotransmitters until the subsequent discovery of bacterial counterparts. While the fundamental aspects of bacterial gasotransmitters remain undefined and necessitate further research, we will discuss a known specific role they play in defense against antibiotics. Considering the current dilemma of multidrug-resistant bacteria we consider it particularly prudent to exploring novel targets and approaches, of which the bacterial gasotransmitters, nitric oxide and hydrogen sulfide represent.
PMID: 25078319
ISSN: 1369-5274
CID: 1440692

The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

Vera, Maria; Pani, Bibhusita; Griffiths, Lowri A; Muchardt, Christian; Abbott, Catherine M; Singer, Robert H; Nudler, Evgeny
Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3'UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.
PMCID:4164936
PMID: 25233275
ISSN: 2050-084x
CID: 1252382

Role of H2S and NO in Bacillus anthracis spore formation and virulence [Meeting Abstract]

Shatalin, K; Nudler, E
Many prokaryotic species generate hydrogen sulfide (H2S) and nitric oxide (NO) enzymatically, from cysteine and arginine, respectively, in their natural environments. Both gases are small freely diffusible signaling molecules that are known to be involved in numerous physiological and pathological processes in mammals. However the biochemistry and physiological role of these gases in bacteria remains largely unknown. We have shown that inactivation of H2S producing enzymes (cystathionine beta-synthase, cystathionine gamma lyase, or 3-mercaptopyruvate sulfurtransferase) and NO-synthase in several Gram (+) and Gram (-) bacteria render them highly sensitive to different classes of antibiotics (Gusarov et al., Science 325 (2009) 1380-1384; Shatalin et al. Science 334 (2011) 986-990). We also presented evidence that Bacillus anthracis-derived NO is critical at the early stage of infection (Shatalin et al. PNAS 105 (2008) 1009-1013). Here we show that: (1) cbs/cse and nos mutations change Bacilli global gene transcription profile; (2) apore formation process in cbs/cse and nos mutants ofB. anthracis is affected; (3) virulence of cbs/cse and nos mutants of B. anthracis is diminished. These results demonstrate that bacterial H2S and NO are an important virulence factors, and that enzymes generated these gases may serve as an attractive target for antimicrobial therapy
EMBASE:71558282
ISSN: 1089-8603
CID: 1154012

UvrD facilitates DNA repair by pulling RNA polymerase backwards

Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny
UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes.
PMCID:4471481
PMID: 24402227
ISSN: 0028-0836
CID: 741112

Therapeutic effect of exogenous hsp70 in mouse models of Alzheimer's disease

Bobkova, Natalia V; Garbuz, David G; Nesterova, Inna; Medvinskaya, Natalia; Samokhin, Alexander; Alexandrova, Irina; Yashin, Valerii; Karpov, Vadim; Kukharsky, Michail S; Ninkina, Natalia N; Smirnov, Andrey A; Nudler, Evgeny; Evgen'ev, Michael
Brain deterioration resulting from "protein folding" diseases, such as the Alzheimer's disease (AD), is one of the leading causes of morbidity and mortality in the aging human population. Heat shock proteins (Hsps) constitute the major cellular quality control system for proteins that mitigates the pathological burden of neurotoxic protein fibrils and aggregates. However, the therapeutic effect of Hsps has not been tested in a relevant setting. Here we report the dramatic neuroprotective effect of recombinant human Hsp70 in the bilateral olfactory bulbectomy model (OBX mice) and 5XFAD mouse models of neurodegeneration. We show that intranasally-administered Hsp70 rapidly enters the afflicted brain regions and mitigates multiple AD-like morphological and cognitive abnormalities observed in model animals. In particular, in both cases it normalizes the density of neurons in the hippocampus and cortex which correlates with the diminished accumulation of amyloid-beta (Abeta) peptide and, in the case of 5XFAD mice, reduces Abeta plaque formation. Consistently, Hsp70 treatment also protects spatial memory in OBX and 5XFAD mice. These studies demonstrate that exogenous Hsp70 may be a practical therapeutic agent for treatment of neurodegenerative diseases associated with abnormal protein biogenesis and cognitive disturbances, such as AD, for which neuroprotective therapy is urgently needed.
PMID: 23985416
ISSN: 1387-2877
CID: 687662

Transcription-replication encounters, consequences and genomic instability

Helmrich, Anne; Ballarino, Monica; Nudler, Evgeny; Tora, Laszlo
To ensure accurate duplication of genetic material, the replication fork must overcome numerous natural obstacles on its way, including transcription complexes engaged along the same template. Here we review the various levels of interdependence between transcription and replication processes and how different types of encounters between RNA- and DNA-polymerase complexes may result in clashes of those machineries on the DNA template and thus increase genomic instability. In addition, we summarize strategies evolved in bacteria and eukaryotes to minimize the consequences of collisions, including R-loop formation and topological stresses.
PMID: 23552296
ISSN: 1545-9985
CID: 306252

RNA polymerase and the ribosome: the close relationship

McGary, Katelyn; Nudler, Evgeny
In bacteria transcription and translation are linked in time and space. When coupled to RNA polymerase (RNAP), the translating ribosome ensures transcriptional processivity by preventing RNAP backtracking. Recent advances in the field have characterized important linker proteins that bridge the gap between transcription and translation: In particular, the NusE(S10):NusG complex and the NusG homolog, RfaH. The direct link between the moving ribosome and RNAP provides a basis for maintaining genomic integrity while enabling efficient transcription and timely translation of various genes within the bacterial cell.
PMCID:4066815
PMID: 23433801
ISSN: 1369-5274
CID: 335482

Methicillin-resistant Staphylococcus aureus Bacterial Nitric-oxide Synthase Affects Antibiotic Sensitivity and Skin Abscess Development

van Sorge, Nina M; Beasley, Federico C; Gusarov, Ivan; Gonzalez, David J; von Kockritz-Blickwede, Maren; Anik, Sabina; Borkowski, Andrew W; Dorrestein, Pieter C; Nudler, Evgeny; Nizet, Victor
Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from l-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.
PMCID:3585076
PMID: 23322784
ISSN: 0021-9258
CID: 231692