Faculty Bibliography

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 (OBE) mouse model. We show that intranasally administered Hsp70 rapidly enters the afflicted brain regions and mitigates multiple AD-like morphological and cognitive abnormalities observed in OBE animals. In particular, it normalized the density of neurons in the hippocampus, which correlated with the diminished accumulation of amyloid b (Ab) peptide. Consistently, Hsp70 also fully protected spatial memory, which remained at the level of control animals for at least eight months following treatment. The long-lasting therapeutic effect of Hsp70 suggests a novel mechanism of action and establishes it as a practical and potent agent for treatment of neurodegenerative diseases associated with abnormal protein biogenesis and cognitive disturbances, such as AD, for which neuroprotective therapy is urgently needed

Heat shock protein 70 (Hsp70) is a molecular chaperone providing tolerance to heat and other challenges at the cellular and organismal levels. We sequenced a genomic cluster containing three hsp70 family genes linked with major histocompatibility complex (MHC) class III region from an extremely heat tolerant animal, camel (Camelus dromedarius). Two hsp70 family genes comprising the cluster contain heat shock elements (HSEs), while the third gene lacks HSEs and should not be induced by heat shock. Comparison of the camel hsp70 cluster with the corresponding regions from several mammalian species revealed similar organization of genes forming the cluster. Specifically, the two heat inducible hsp70 genes are arranged in tandem, while the third constitutively expressed hsp70 family member is present in inverted orientation. Comparison of regulatory regions of hsp70 genes from camel and other mammals demonstrates that transcription factor matches with highest significance are located in the highly conserved 250-bp upstream region and correspond to HSEs followed by NF-Y and Sp1 binding sites. The high degree of sequence conservation leaves little room for putative camel-specific regulatory elements. Surprisingly, RT-PCR and 5'/3'-RACE analysis demonstrated that all three hsp70 genes are expressed in camel's muscle and blood cells not only after heat shock, but under normal physiological conditions as well, and may account for tolerance of camel cells to extreme environmental conditions. A high degree of evolutionary conservation observed for the hsp70 cluster always linked with MHC locus in mammals suggests an important role of such organization for coordinated functioning of these vital genes

During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that translates the newly synthesized transcript. Our in vivo measurements show that the overall elongation rate of transcription is tightly controlled by the rate of translation. Acceleration and deceleration of a ribosome result in corresponding changes in the speed of RNAP. Moreover, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. The stimulating effect of a ribosome on RNAP is achieved by preventing its spontaneous backtracking, which enhances the pace and also facilitates readthrough of roadblocks in vivo. Such a cooperative mechanism ensures that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria. Although Rho was the first termination factor to be discovered, the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle; that is, it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP-Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allosterically induced rearrangement of the RNAP catalytic centre by means of the evolutionarily conserved mobile trigger-loop domain, which is also required for EC dissociation. The key structural and functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution

Multi-subunit DNA-dependent RNA polymerases synthesize RNA molecules thousands of nucleotides long. The reiterative reaction of nucleotide condensation occurs at rates of tens of nucleotides per second, invariably linked to the translocation of the enzyme along the DNA template, or threading of the DNA and the nascent RNA molecule through the enzyme. Reiteration of the nucleotide addition/translocation cycle without dissociation from the DNA and RNA requires both isomorphic and metamorphic conformational flexibility of a magnitude substantial enough to accommodate the requisite molecular motions. Here we review some of the more recently acquired insights into the structural flexibility and morphic fluctuations of RNA polymerases and their mechanistic implications

Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms. NO-mediated resistance is achieved through both the chemical modification of toxic compounds and the alleviation of the oxidative stress imposed by many antibiotics. Our results suggest that the inhibition of NOS activity may increase the effectiveness of antimicrobial therapy

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bgamma, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bgamma. We also show that infection by L. pneumophila leads to eEF1A-mediated activation of the heat shock regulatory protein HSF1 in a virulence-dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila-infected cells.

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx--a desmethyl derivative of myxopyronin B--complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs

Natural RNA sensors of small molecules (a.k.a. riboswitches) regulate numerous metabolic genes. In bacteria, these RNA elements control transcription termination and translation initiation by changing the folding pathway of nascent RNA upon direct binding of a metabolite. To identify and study riboswitches we used in vitro reconstituted solid-phase transcription elongation/termination system. This approach allows for direct monitoring of ligand binding and riboswitch functioning, establishing the working concentration of a ligand as a function of RNA polymerase speed, and also probing RNA structure of the riboswitch. Using this system we have been able to identify and characterize first several riboswitches including those involved in vitamin biosynthesis and sulfur metabolism. The system can be utilized to facilitate biochemical studies of riboswitches in general, i.e., to simplify analysis of riboswitches that are not necessarily involved in transcriptional control

The heat shock (HS) response is the major cellular defense mechanism against acute exposure to environmental stresses. The hallmark of the HS response, which is conserved in all eukaryotes, is the rapid and massive induction of expression of a set of cytoprotective genes. Most of the induction occurs at the level of transcription. The master regulator, heat shock transcription factor (HSF, or HSF1 in vertebrates), is responsible for the induction of HS gene transcription in response to elevated temperature. Under normal conditions HSF is present in the cell as an inactive monomer. During HS, HSF trimerizes and binds to a consensus sequence in the promoter of HS genes, stimulating their transcription by up to 200-fold. We have shown that a large, noncoding RNA, HSR1, and the translation elongation factor eEF1A form a complex with HSF during HS and are required for its activation