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Duennwald M.L.,Boston Biomedical Research Institute | Echeverria A.,Boston Biomedical Research Institute | Shorter J.,University of Pennsylvania
PLoS Biology | Year: 2012

How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders. © 2012 Duennwald et al.


Komaba S.,Boston Biomedical Research Institute | Coluccio L.M.,Boston Biomedical Research Institute
Journal of Biological Chemistry | Year: 2010

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that fulllength rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3), two phosphoinositides that play important roles in cell signaling. Binding is not Ca2+-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP2 and PIP3 in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP2 localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Albert Wang C.-L.,Boston Biomedical Research Institute | Coluccio L.M.,Boston Biomedical Research Institute
International Review of Cell and Molecular Biology | Year: 2010

The actin cytoskeleton is regulated by a variety of actin-binding proteins including those constituting the tropomyosin family. Tropomyosins are coiled-coil dimers that bind along the length of actin filaments. In muscles, tropomyosin regulates the interaction of actin-containing thin filaments with myosin-containing thick filaments to allow contraction. In nonmuscle cells where multiple tropomyosin isoforms are expressed, tropomyosins participate in a number of cellular events involving the cytoskeleton. This chapter reviews the current state of the literature regarding tropomyosin structure and function and discusses the evidence that tropomyosins play a role in regulating actin assembly. © 2010 Elsevier Inc.


Duennwald M.L.,Boston Biomedical Research Institute
Methods | Year: 2011

Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast. © 2010 Elsevier Inc.


Kitazawa T.,Boston Biomedical Research Institute | Kitazawa K.,Boston Biomedical Research Institute
Journal of Physiology | Year: 2012

Each segment along arterial vessels adapts to different circumstances, including blood pressure and sympathetic innervation. PKC and Rho-associated kinase (ROCK) Ca2+-sensitizing pathways leading to myosin phosphatase inhibition are critically involved in α1-adrenoceptor-mediated vascular smooth muscle contraction in distinctive time-dependent manners. We tested whether the amplitude and time course of each pathway varies dynamically between arterial segments. Using pharmacological approaches, we determined the time-dependent roles of Ca2+ release, Ca2+ influx, PKC and ROCK in α1-agonist-induced contraction and phosphorylation of key proteins in denuded rat small mesenteric artery, midsized caudal artery and thoracic aorta. SR Ca2+ release and voltage-dependent Ca2+ influx were essential for the initial rising and late sustained phases, respectively, of phenylephrine-induced contraction, regardless of arterial size. In small mesenteric arteries, α1A-subtype-specific antagonists and inhibitors of PKC, but not ROCK, markedly reduced the initial and late phases of contraction in a non-additive manner and suppressed phosphorylation of myosin light chain (MLC) and CPI-17, but not myosin targeting subunit of myosin light chain phosphatase (MYPT1). In aorta, an α1D-specific antagonist reduced both the initial and late phases of contraction with a significant decrease in MLC but not CPI-17 or MYPT1 phosphorylation. ROCK inhibitors, but not PKC inhibitors, suppressed the sustained phase of contraction with a decrease in MLC and MYPT1 phosphorylation in the aorta. The effect of ROCK inhibitors was additive with the α1D-antagonist. The results for midsized arteries were intermediate. Thus, the PKC-CPI-17 Ca2+-sensitizing pathway, which is dependent on PKC subtype and a Ca2+-handling mechanism, and is downstream of α1A receptors, plays a major role in α1-agonist-induced contraction of small resistance arteries in the splanchnic vascular beds. The effect of PKC and ROCK increases and decreases, respectively, with decreasing arterial size. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.


Sarkes D.,Boston Biomedical Research Institute | Rameh L.E.,Boston Biomedical Research Institute
Biochemical Journal | Year: 2010

PtdIns5P was discovered in 1997 [Rameh, Tolias, Duckworth and Cantley (1997) Nature 390, 192-196], but still very little is known about its regulation and function. Hitherto, studies of PtdIns5P regulation have been hindered by the inability to measure cellular PtdIns5P using conventional HPLC, owing to poor separation from PtdIns4P. In the present paper we describe a new HPLC method for resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of basal and inducible levels of cellular PtdIns5P in the context of other phosphoinositides. Using this new method, we found that PtdIns5P is constitutively present in all cells examined (epithelial cells, fibroblasts and myoblasts, among others) at levels typically 1-2% of PtdIns4P levels. In the β-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than inmost cells (2.5-4% of PtdIns4P). Using subcellular fractionation, we found that the majority of the basal PtdIns5P is present in the plasma membrane, but it is also enriched in intracellular membrane compartments, especially in SER (smooth endoplasmic reticulum) and/or Golgi, where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was also found in fractions containing very-low-density vesicles. Knockdown of PIP4K (PtdIns5P 4-kinase) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, whereas treatment with Brefeldin A results in a subtle, but reproducible, change in PtdIns5P distribution. These results indicate that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P2 synthesis may play a role in Golgi-mediated vesicle trafficking. © The Authors.


Duennwald M.L.,Boston Biomedical Research Institute
Prion | Year: 2011

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment. © 2011 Landes Bioscience.


Huh Y.H.,Boston Biomedical Research Institute | Sherley J.L.,Boston Biomedical Research Institute
Stem Cells | Year: 2011

Although nonrandom sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying nonrandom segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify nonrandom segregation. During nonrandom segregation, at each mitosis, asymmetrically selfrenewing DSCs continuously cosegregate to themselves the set of chromosomes that contain immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing nonrandom segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC-specific properties and ex vivo-expanded mouse hair follicle stem cells, the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well but is cloaked from immunodetection, and the cloaking entity is acid labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for nonrandom segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for nonrandom sister chromatid segregation in DSCs. © AlphaMed Press.


Patent
Boston Biomedical Research Institute | Date: 2013-04-11

Disclosed herein are compositions and methods useful for controlling -amyloid levels. In particular, the instant invention relates to an antibody that catalyzes hydrolysis of -amyloid at a predetermined amide linkage are provided. The present invention also provides a vectorized antibody that is capable of crossing the blood brain barrier and is also capable of catalyzing the hydrolysis of -amyloid at a predetermined amide linkage. Also provided are methods for modulating -amyloid levels in vivo using antibodies that bind to -amyloid. These compositions and methods have therapeutic applications, including the treatment of Alzheimers disease.


Patent
Boston Biomedical Research Institute | Date: 2015-04-16

Disclosed herein are compositions and methods useful for controlling -amyloid levels. In particular, the instant invention relates to an antibody that catalyzes hydrolysis of -amyloid at a predetermined amide linkage are provided. The present invention also provides a vectorized antibody that is capable of crossing the blood brain barrier and is also capable of catalyzing the hydrolysis of -amyloid at a predetermined amide linkage. Also provided are methods for modulating -amyloid levels in vivo using antibodies that bind to -amyloid. These compositions and methods have therapeutic applications, including the treatment of Alzheimers disease.

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