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Pathologic α-syn destabilizes the TSC 1 and 2 complex leading to mTORC1 activation, enhanced protein translation and neurodegeneration in PD.Abstract:Pathological α-synuclein (α-syn) plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson’s disease (PD). Disruption of protein homeostasis is thought be central to PD pathogenesis, however the molecular mechanism of this deregulation is poorly understood. Here we report that pathologic α-syn binds to tuberous sclerosis protein (TSC) 2 and destabilizes the TSC1-TSC2 complex leading to activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and enhanced mRNA translation. Dopamine neuron loss, behavioral deficits and aberrant biochemical signaling in the α-syn preformed fibril (PFF) and Drosophila α-syn transgenic models of pathologic α-syn induced degeneration were attenuated by genetic and pharmacologic inhibition of mTOR and protein translation. Our findings establish a potential molecular mechanism by which pathologic α-syn activates mTORC1 leading to enhanced protein translation and concomitant neurodegeneration in PD.

AMPA-selective glutamate receptors mediate the transduction of signals between the neuronal circuits of the hippocampus1. The trafficking, localization, kinetics and pharmacology of AMPA receptors are tuned by an ensemble of auxiliary protein subunits, which are integral membrane proteins that associate with the receptor to yield bona fide receptor signalling complexes2. Thus far, extensive studies of recombinant AMPA receptor-auxiliary subunit complexes using engineered protein constructs have not been able to faithfully elucidate the molecular architecture of hippocampal AMPA receptor complexes. Here we obtain mouse hippocampal, calcium-impermeable AMPA receptor complexes using immunoaffinity purification and use single-molecule fluorescence and cryo-electron microscopy experiments to elucidate three major AMPA receptor-auxiliary subunit complexes. The GluA1-GluA2, GluA1-GluA2-GluA3 and GluA2-GluA3 receptors are the predominant assemblies, with the auxiliary subunits TARP-γ8 and CNIH2-SynDIG4 non-stochastically positioned at the B'/D' and A'/C' positions, respectively. We further demonstrate how the receptor-TARP-γ8 stoichiometry explains the mechanism of and submaximal inhibition by a clinically relevant, brain-region-specific allosteric inhibitor.

RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch., Many RNAs become functional before their synthesis completes. Here the authors employ a single-molecule vectorial folding assay mimicking RNA transcription and show that the ZTP riboswitch is kinetically controlled and activated by slower unwinding and strategic pausing.

In eukaryotes, the origin recognition complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for prereplication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression after pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient MMR. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S phase.

A human telomere ends in a single-stranded 3′ tail, composed of repeats of T2AG3. G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.

The circumsporozoite protein (CSP) forms a dense coat on the surface of the sporozoite, the infective stage of the malaria parasite. The central repeat region of CSP is a critical component of the only licensed malaria vaccine yet little is known about its structure or function. We found that sporozoite mutants with severely truncated or scrambled repeats have impaired motility due to altered adhesion site formation and dynamics, suggesting that the CSP repeats provide a cohesive environment in which adhesion sites can form. We hypothesized that biophysical properties of the repeats are important in this role and interrogated this using single-molecule fluorescence-force spectroscopy. We show that the repeats are a stiff, linear spring with elastic properties, dependent upon length and lost when the repeats are scrambled. These data are the first evidence that the CSP repeat region serves a functional role during infection and motility, likely mediated through its biophysical properties.SummaryNo clear function of the central repeat region of the malaria circumsporozoite protein has been described to date, despite its central role in the only licensed malaria vaccine. Here we use mutational analysis and single-molecule fluorescence-force spectroscopy to describe the structural properties and determine the function of this conserved region and important vaccine target.HighlightsThe CSP repeats have properties of a linear springScrambling or large truncations of the repeats leads to defects in sporozoite motilityMotility defects are attributed to abnormal formation of adhesion sites

In eukaryotes, the Origin Recognition Complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for pre-replication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression post pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S-phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutS and enhances the chromatin-association of MutL, thus enabling efficient mismatch repair. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S-phase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/443400v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@bfdaa8org.highwire.dtl.DTLVardef@1ac791dorg.highwire.dtl.DTLVardef@436d1corg.highwire.dtl.DTLVardef@b07689_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHuman Orc6 is dispensable for G1 licensing, but required for S-phase progression C_LIO_LIHuman Orc6 at the replication fork is an accessory factor for MMR complex C_LIO_LIDepletion of hOrc6 sensitizes cells to DNA damage and impairs ATR activation C_LIO_LIHuman Orc6 regulates MMR complex assembly and activity C_LI

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain–mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

SummaryRiboswitches function through cotranscriptional conformation switching governed by cognate ligand concentration, RNA folding and transcription elongation kinetics. To investigate how these parameters influence riboswitch folding, we developed a novel vectorial folding assay (VF) in which the superhelicase Rep-X sequentially liberates the RNA strand from a heteroduplex in a 5’-to-3’ direction, mimicking the nascent chain emergence during transcription. The RNA polymerase (RNAP)-free VF recapitulates the kinetically controlled cotranscriptional folding of a ZTP riboswitch, whose activation is favored by slower transcription, strategic pausing, or a weakened transcriptional terminator. New methods to observe positions and local rates of individual helicases show an average Rep-X unwinding rate similar to bacterial RNAP elongation (~60 nt/s). Real-time single-molecule monitoring captured folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These methods allow observation of individual folding RNAs as they occupy distinct folding channels within the landscape that controls gene expression and showed that riboswitch fate control is encoded in its sequence and is readily interpreted by a directionally moving protein even in the absence of an RNA polymerase.

Tandem repeats of guanine rich sequences are ubiquitous in the eukaryotic genome. For example, in the human cells, telomeres at the chromosomal ends comprise of kilobases repeats of T2AG3. Four such repeats can form G-quadruplexes (GQs). Biophysical studies have shown that GQs formed from four consecutive repeats possess high diversity both in their structure and in their response to tension. In principle, a GQ can form from any four repeats that may not even be consecutive. In order to investigate the dynamics of GQ possessing such positional multiplicity, we studied five and six repeats human telomeric sequence using single molecule FRET as well as its combination with optical tweezers. Our results suggest preferential formation of GQs at the 3’ end both in K+ and Na+ solutions although minority populations with a 5’ GQ or long-loop GQs were also observed. Using a vectorial folding assay which mimics the directional nature of telomere extension, we found that the 3’ preference holds even when folding is allowed to begin from the 5’ side. Interestingly, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. Location of GQ on a long G-rich telomeric overhang and reduction in diversity of GQ conformations and mechanical responses through adjacent sequences have important implications in processes such as telomerase inhibition, alternative lengthening of telomeres, T-loop formation, telomere end protection and replication.

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K(+) in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na(+), although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

A one-pot, economically viable ‘green’ synthesis of silver glyconanoparticles is reported with sugar cane juice. The morphology and size of the particles could be tuned by changing reaction conditions like the temperature, pH, concentration etc. Sugar and non-sugar biomolecules in sugar cane acted as reducing and/or capping agents in the synthesis. These glyconanoparticles exhibited exceptional photoluminescence and ferromagnetism at room temperature. This general route can be employed for the synthesis of other noble metal nanoparticles. Annealing of silver nanoparticles on underlying polymer fibers yielded unprecedented hollow helical structures as opposed to rods obtained on silicon wafers. A surface-assisted self-assembly mechanism has been proposed to explain the peculiar morphology of annealed structures. Photoluminescence of the as-annealed structures has also been demonstrated. These hollow and solid rods have potential applications in the fabrication of nano-circuits and optoelectronics.

G-quadruplexes (GQs) are constituted by stacks of G-quartets formed in guanine rich sequences in nucleic acids. Among its many functions, GQ plays a pivotal role in regulating telomere length in cells. Mechanistic understanding of dynamics of GQ is key to understanding its biological functions. Integrated single molecule force-fluorescence spectroscopy has been used herein to study the dynamics of human telomeric GQs under tension. GQ unfolding at physiological K+ concentration showed three features: a significant population resisted perturbation by force (∼35 pN) and remained in the folded state, while unfolding culminated into either a partially or completely unfolded state. Complete unfolding of GQs occurred stochastically within a wide spectrum of forces between 3 and 35 pN, even within the same molecule across different stretching cycles. Correspondingly, although GQs exhibited diverse refolding behavior, most of the refolding occurred within 5-10 pN. Such diversity in the forces required for complete unfolding of GQs was also observed with Na+. Replacement of a single guanine precluded GQ formation at lower metal ion concentrations (up to 100 mM) and only partially folded GQ-like species were induced which unfolded/refolded at lower forces with no evident hysteresis. At high metal ion concentrations (1M of Na+/K+) GQ with single point mutation showed an unfolding/refolding behavior redolent of GQ, suggesting formation of GQ-like species. The understanding of different levels of stability in human telomeric GQ can be leveraged into developing anti-cancer drugs.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G 1 -S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G 1 -S transition, possibly providing an intracellular milieu favorable for its replication.

Silica fibers are of technological significance in view of their applications in sensing, photocatalysis, filtration, biological scaffolds, photoluminescence, composites, etc. A combination of electrospinning and sol–gel technique has been employed to synthesize inorganic or ceramic sub-micron fibers starting from a novel oligomeric pre-ceramic precursor, Poly(methylhydrosiloxane) (PMHS). The composite fibers were calcined at temperatures in the range of 550 °C to 1400 °C to obtain slica fibers. The electrospun pre-ceramic fibers and derived silica sub-micron fibers were characterized by FESEM, TEM, XRD, Raman spectroscopy and FTIR. Calcination transformed the smooth pre-ceramic fibers to compact silica fibers that consist of nano-sized silica particles. Calcinations at lower temperatures yielded more amorphous silica fibers, but calcination at 1400 °C yielded a polymorph of crystalline silica, α-cristobalite. Building up of an ordered Si–O network with increased temperature was verified by Infra-Red and Raman Spectroscopy. Photoluminescence (PL) study of the electrospun fibers showed their potential as blue, green and red light emitters at room temperature at different excitation wavelengths, and a blue/violet emission with the UV excitation not seen previously. Although calcination of PMHS fibers decreased PL intensity, a close correspondence in both the emission and excitation PL spectra along with the PL life times of differently calcined fibers is probably owing to the presence of similar defect centers. Tuning of PL should allow potential applications in optoelectronics, medical nanoprobes and bio-labeling and sensing.

The fabrication of tissue engineering scaffolds necessitates amalgamation of a multitude of attributes including a desirable porosity to encourage vascular invasion, desired surface chemistry for controlled deposition of calcium phosphate-based mineral as well as ability to support attachment, proliferation, and differentiation of lineage specific progenitor cells. Scaffold fabrication often includes additional surface treatments to bring about desired changes in the surface chemistry. In this perspective, this review documents the important natural and synthetic scaffolds fabricated for bone tissue engineering applications in tandem with the surface treatment techniques to maneuver the biocompatibility of engineered scaffolds. This review begins with a discussion on the fundamental concepts related to biocompatibility as well as the characteristics of the biological micro-environment. The primary focus is to discuss the effects of surface micro/nano patterning on the modulation of bone cell response. Apart from reviewing a host of experimental studies reporting the functionality of osteoblast-like bone cells and stem cells on surface modified or textured bioceramic/biopolymer scaffolds, theoretical insights to predict cell behavior on a scaffold with different topographical features are also briefly analyzed.