Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity.

The Hsp90 molecular chaperones are ATP-dependent enzymes that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotes have organelle-specific Hsp90 paralogs that are adapted to each subcellular environment. The mitochondrial Hsp90, TNF receptor-associated protein 1 (TRAP1), supports the folding and activity of electron transport components and is increasingly appreciated as a critical player in mitochondrial signaling. Calcium plays a well-known and important regulatory role in mitochondria where it can accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we found here that calcium can replace magnesium, the essential enzymatic cofactor, to support TRAP1 ATPase activity. Anomalous X-ray diffraction experiments revealed a calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP α-phosphate and completely distinct from the magnesium-binding site adjacent to the β- and γ-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, was noncooperative, whereas calcium binding resulted in cooperative hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggested a mechanism for this cooperative behavior. Because of the cooperativity, at high ATP concentrations, ATPase activity was higher with calcium, whereas the converse was observed at low ATP concentrations. Integrating these observations, we propose a model in which the divalent cation choice can control switching between noncooperative and cooperative TRAP1 ATPase mechanisms in response to varying ATP concentrations. This switching may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle and its consequent effects on different mitochondrial clients

A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity.

Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. In this study, we address the regulatory function of this extension or 'strap' and demonstrate its responsibility for an unusual temperature dependence in ATPase rates. This dependence is a consequence of a thermally sensitive kinetic barrier between the apo 'open' and ATP-bound 'closed' conformations. The strap stabilizes the closed state through trans-protomer interactions. Displacement of cis-protomer contacts from the apo state is rate-limiting for closure and ATP hydrolysis. Strap release is coupled to rotation of the N-terminal domain and dynamics of the nucleotide binding pocket lid. The strap is conserved in higher eukaryotes but absent from yeast and prokaryotes suggesting its role as a thermal and kinetic regulator, adapting Hsp90s to the demands of unique cellular and organismal environments

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context

Composição química e atividade antifúngica do óleo essencial de Zanthoxylum petiolare A. St. -Hil. & Tul (RUTACEAE) / Chemical composition and antifungal activity of the essential oil of Zanthoxylum petiolare A. St.-Hil. & Tul (RUTACEAE)

O objetivo deste trabalho foi avaliar o perfil químico e a atividade antifúngica do óleo essencial das folhas da espécie Zanthoxylum petiolare A.St.-Hil. & Tul. (Rutaceae). Para isso foram realizados estudo fitoquímico e antifúngico. A extração do óleo foi realizada utilizando aparelho Clevenger e a composição química foi determinada por Cromatografia Gasosa acoplada a Espectrometria de Massas (CG-EM). Os testes de sensibilidade foram realizados por microdiluição em caldo em cepas de fungos dermatófitos e leveduriformes. A análise do óleo essencial identificou 20 compostos químicos, onde o espatulenol (19,85%), geranial (16,31%), cis-Citral (12,54%) foram compostos majoritário. O óleo essencial mostrou atividade antifúngica contra as cepas de Trichophytom rubrum, apresentando 0, 156 mg/ml e 0,312 mg/ml para CIM e CFM, respectivamente, não manifestando ação contra Candida spp. Este é o primeiro estudo a investigar a atividade antifúngica de Z. petiolare que se mostrou uma espécie promissora para estudos posteriores com o propósito de se identificar novos metabólitos com ações farmacológicas

Asymmetric Mechanism of Molecular Chaperone Hsp90

Hsp90 is a highly conserved, ATP-dependent molecular chaperone that is essential for maintaining the functions of its client proteins. It has been estimated that about 10% of the proteome of a eukaryotic cell interacts with Hsp90. A large subset of this portion consists of protein kinases and steroid hormone receptors, putting Hsp90 as the master regulator of many essential cellular functions. The mechanism of how Hsp90 uses ATP hydrolysis to carry out its function remains unclear. Structural studies of Hsp90 revealed that Hsp90 is a V-shaped homodimer with each protomer composed of three well-folded domains: an ATP-binding N-terminal Domain (NTD), a Middle Domain (MD), and a C-terminal dimerization Domain (CTD). Efficient ATP hydrolysis by Hsp90 requires that the dimer forms a closed state where NTDs are dimerized, forming a closed ”clamp” conformation that is stabilized by ATP binding. The kinetics of forming this stably closed state is not driven by ATP binding, as there are other rate-limiting steps that need to occur within the protein to allow the NTDs to be dimerized. So how does Hsp90 actually use the energy from ATP to remodel its client protein? the focus of this thesis examines the other possibility of this happening during ATP hydrolysis. In the first chapter, I followed up an observation made by a previous graduate student Laura Lavery. She observed that the ATP-bound closed state of a mitochondrial Hsp90 (TRAP1) is asymmetric. The asymmetry is most prominent at the juncture between the MD:CTD interface—one protomer is buckled while the other remains straight, resembling the same conformation previously observed in the symmetric closed ”clamp” state. This buckling happens precisely where conserved binding sites have been mapped for client proteins. This suggests that if conformational changes were to occur due to ATP hydrolysis, subsequent rearrangements of the asymmetric MD:CTD interfaces back to the previously observed symmetric closed state vii could be used to drive client protein remodeling. Using a combination of biophysical methods (crystallography, Double Electron-Electron Resonance (DEER), and FRET), we observed that TRAP1 hydrolyzes the 2 bound ATPs sequentially. The buckled protomer hydrolyzes the first ATP, which is then followed by a flip in the asymmetry (the buckled conformation becomes straight and vice versa, on each side), which primes the second ATP for hydrolysis by a buckled protomer. In this model, the MD:CTD interface is guaranteed to undergo remodeling with each ATP hydrolysis and would make efficient use of energy from ATP. The implications for this asymmetric ATP hydrolysis mechanism may also be relevant to other Hsp90s. While we have not observed any other asymmetric Hsp90 structures by itself, several functional Hsp90 complexes seen so far seem to have asymmetric composition/arrangements of their components. In the second chapter, we explore how TRAP1 ATPase activity can be modulated by different divalent cations as co-factors. Despite having two ATP binding sites, the ATPase activity of most Hsp90 homologs appears to be non-cooperative (each site behaves independently from one another). However, we saw that ATPase activity of TRAP1 can be cooperative in presence of calcium, and the activity in presence of magnesium appears to be bi-phasic, with higher activity at low ATP concentrations. This unique behavior of TRAP1 may yet be another adaptation of the Hsp90 machine that has evolved within the mitochondrial matrix environment. Using crystallography, we also discovered that calcium binds to the NTD of TRAP1 unlike previously observed chaperone/calcium/ATP complexes. While the exact biological role for this phenomena is not yet clear, these findings provide a clear molecular basis for the regulation of TRAP1 by calcium. Taken together, the work described in this thesis provide insights into the mechanism of ATP hydrolysis by Hsp90, and a potential role that TRAP1 plays in calcium/magnesium-regulated mitochondrial physiology

Long-Range Structural Changes in the Meiotic Nucleus Revealed by Changes in Stress Communication Along the Chromosome

Homologous recombination drives structural reorganization of the nucleus in early meiosis. In order to investigate the connection between homolog pairing, meiotic progression, and the dynamics of the underlying chromatin, we tracked flourescently labeled homolog pairs in synchronized S. cerevisae. Various previously unreported statistics of the anomalous inter-loci motion correlate with meiotic progression and can be quantitatively reproduced by a simple polymer model of the sister chromatids. The first of these is the distribution of waiting times for the homologous loci to come into and out of contact with each other (loosely, inter-locus “looping” and “unlooping” times). The full shape of the looping time distribution can be quantitatively reproduced by a simple model of two polymers diffusing independently in a spherical confinement. This finding suggests a dominant role for diffusion-limited, undirected search in homolog pairing in early meiosis. This is in sharp contrast with the intuition that a heavy-tailed search process could never drive such a critical cell-cycle stage. We further show that the inter-locus velocity-velocity correlation (VVC) quantitatively matches analytical results for the inter-locus VVC of our polymer model, allowing us to leverage our analytical theory to extract the time scale of stress communication between the labeled loci along the chromosome. We show that stresses can take tens of minutes to propagate between loci on paired chromosomes, and that the increasing connectivity between the chromosomes as the cell progresses through meiosis can be quantified by the shortening of this communication time. Our study highlights the power of coarse-grained polymer models to analyze dynamic structural properties of the nucleus in vivo and the importance of analytical theory for uncovering intracellular connections that might be obscured by lag times of many minutes

Long-Range Structural Changes in the Meiotic Nucleus Revealed by Changes in Stress Communication Along the Chromosome

Homologous recombination drives structural reorganization of the nucleus in early meiosis. In order to investigate the connection between homolog pairing, meiotic progression, and the dynamics of the underlying chromatin, we tracked flourescently labeled homolog pairs in synchronized S. cerevisae. Various previously unreported statistics of the anomalous inter-loci motion correlate with meiotic progression and can be quantitatively reproduced by a simple polymer model of the sister chromatids. The first of these is the distribution of waiting times for the homologous loci to come into and out of contact with each other (loosely, inter-locus “looping” and “unlooping” times). The full shape of the looping time distribution can be quantitatively reproduced by a simple model of two polymers diffusing independently in a spherical confinement. This finding suggests a dominant role for diffusion-limited, undirected search in homolog pairing in early meiosis. This is in sharp contrast with the intuition that a heavy-tailed search process could never drive such a critical cell-cycle stage. We further show that the inter-locus velocity-velocity correlation (VVC) quantitatively matches analytical results for the inter-locus VVC of our polymer model, allowing us to leverage our analytical theory to extract the time scale of stress communication between the labeled loci along the chromosome. We show that stresses can take tens of minutes to propagate between loci on paired chromosomes, and that the increasing connectivity between the chromosomes as the cell progresses through meiosis can be quantified by the shortening of this communication time. Our study highlights the power of coarse-grained polymer models to analyze dynamic structural properties of the nucleus in vivo and the importance of analytical theory for uncovering intracellular connections that might be obscured by lag times of many minutes

A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity.

Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. In this study, we address the regulatory function of this extension or 'strap' and demonstrate its responsibility for an unusual temperature dependence in ATPase rates. This dependence is a consequence of a thermally sensitive kinetic barrier between the apo 'open' and ATP-bound 'closed' conformations. The strap stabilizes the closed state through trans-protomer interactions. Displacement of cis-protomer contacts from the apo state is rate-limiting for closure and ATP hydrolysis. Strap release is coupled to rotation of the N-terminal domain and dynamics of the nucleotide binding pocket lid. The strap is conserved in higher eukaryotes but absent from yeast and prokaryotes suggesting its role as a thermal and kinetic regulator, adapting Hsp90s to the demands of unique cellular and organismal environments