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F Actin Bundling Dynamics and Stiffness of the TRIOBP-4 F Actin Bundle
Tuesday, March 1, 2016 cardiomyocytes and localize into lysosomes. Here, we extended this study following the internalization of full length (FL) and variable domain (VL) AL proteins into human cardiomyocytes (AC16 cell line) by real-time livecell image analysis (IncuCyte ZOOMÔ). Our results show that soluble protein internalizes into AC16 cells in a size-dependent manner, thus VL domains internalizes faster than the FL proteins. Experiments using endocytic inhibitors have shown that soluble proteins internalize by a Clathrindependent endocytic pathway and localize in perinuclear compartments. Further on, we have studied the internalization of preformed amyloid fibrils and their effect when co-incubated with soluble protein. External aggregates rapidly surround the cells, strongly interact with the cell membrane, and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation (cell mediated seeding). Although fibrils are predominantly extracellular, a fraction of them is also internalized. Finally, our attempt to clarify the role of amyloid fibrils in the disease revealed their cytotoxic potential at significant low concentrations compared to soluble AL proteins. This study would help us to understand the unique aspects of behind light chain internalization and cytotoxicity in AL amyloidosis. 1980-Pos Board B124 Insights into the Genesis of Light Chain Amyloid Assembly Pinaki P. Misra, Luis M. Blancas-Mejia, Marina Ramirez-Alvarado. Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. In many amyloid diseases, proteins exhibit a gradual stepwise conversion of monomers to mature amyloid fibrils through an array of intermediate structures with different physicochemical properties. Elucidation of these pathways and structures is especially important as many of these intermediates are reported to be cytotoxic and plays a critical role in the initiation of amyloid assembly. One such amyloid disease caused by deposition of insoluble amyloid fibrils is light-chain (AL) amyloidosis, resulting from plasma cell dyscrasia and invariably leads to cell death and organ failure. Although there is a host of studies conducted on the aggregation of light chain and its mutants, the exact pathway of amyloid nucleation in AL amyloidosis is unclear. Some reports show early oligomeric species during aggregation of the wild type, non amyloidogenic control kI O18/O8 and the amyloidogenic AL-09 variable domain in vivo as well as in presence of certain glycosaminoglycans (GAGs) in vitro. Furthermore the aggregation kinetics of the variable domain (VL) in kI O18/O8 and its mutants has also been reported to show a considerable modulation in aggregation rate in presence of GAGs. In this work, we show small spherical aggregates at early time point of aggregation reaction of kI O18/O8VL, AL-09 VL and AL-103 VL by electron microscopy (EM). Consistent with the physicochemical properties of oligomers, these small spherical aggregate are ThTnegative and act as genesis centers for mature fibrils. Furthermore from our concentration dependence assays, we observed that the aggregation rate is mostly dependent on the dimer dissociation constant of the proteins rather than the involvement of these early spherical aggregates. 1981-Pos Board B125 Mitochondrially-Derived Peptides as Defense Against Amyloid Protein Misfolding Kazuki Teranishi, Alan Okada, Kelvin Yen, Pinchas Cohen, Ralf Langen. USC, los Angeles, CA, USA. The misfolding and aggregation of proteins is the pathological hallmark of amyloid diseases including Alzheimer’s disease (AD), familial amyloid polyneuropathy, and type 2 diabetes mellitus (T2DM). Insofar as the misfolding process results in the formation of pathogenic species, inhibition of misfolding is thought to be a promising approach to the prevention of the aforementioned diseases. Mitochondrial dysfunction has been implicated in many amyloid diseases suggesting an important mitochondrial role. Humanin and its derivatives are a novel class of mitochondrially-derived peptides with insulin sensitizing activity and various cytoprotective effects in AD and other disease model systems. Here we tested whether humanin can promote health in misfolding diseases by acting as a molecular chaperone that prevents amyloid misfolding. We tested whether islet amyloid polypeptide (IAPP), a peptide that misfolds and causes toxicity in T2DM, would be responsive to humanin. Using thioflavin T fluorescence we found that substoichiometric concentrations of the humanin-S14G analogue (HNG) potently inhibit the misfolding of hIAPP. As assessed by circular dichroism, HNG prevents misfolding by maintaining hIAPP in a predominantly random coil structure in solution. Unexpectedly, site-directed spin labeling of HNG in concert with electron paramagnetic resonance (EPR) revealed that HNG is capable of forming higher order complexes. Ongoing investigation into the potency of these higher order structures will test whether they participate in the inhibition of IAPP misfolding. Finally HNG appears to have biological effects including a partial attenuation of IAPP-mediated cytotoxicity and a
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reversal of the increase in oxygen consumption seen when INS832/13 cells, a rodent insulinoma cell line, are challenged with IAPP. In summary our studies suggest the possibility that humanin, and perhaps other mitochondriallyderived peptides, have chaperone-like properties that could reverse the pathological processes involved in the development of T2DM. 1982-Pos Board B126 Biophysics of Tardigrade Survival Samantha Piszkiewicz1, Aakash Mehta1, Thomas Boothby2, Bob Goldstein2,3, Gary Pielak1,4. 1 Chemistry, UNC Chapel Hill, Chapel Hill, NC, USA, 2Biology, UNC Chapel Hill, Chapel Hill, NC, USA, 3Lineberger Comprehensive Cancer Center, UNC Chapel Hill, Chapel Hill, NC, USA, 4Biochemistry and Biophysics, UNC Chapel Hill, Chapel Hill, NC, USA. Tardigrades, commonly known as water bears, are microorganisms that can survive extreme conditions: temperatures from 274 C to 151 C, pressures from 0 to 6,000 atmospheres, 1,000 times more radiation than the average animal, 10 days in space and over 20 years of dehydration. However, little is known about the mechanisms tardigrades use to survive these harsh environments. The Cytoplasmic Abundant Heat Soluble (CAHS) class of proteins is overexpressed by tardigrades under dehydration stress and the CAHS genes are essential for surviving dehydration. We have expressed these proteins in Escherichia coli. Circular dichroism spectropolarimetry and multidimensional, multinuclear NMR spectroscopy data indicate that the purified proteins are intrinsically disordered in dilute solution and form temperature-dependent reversible gel networks at higher concentrations. 1983-Pos Board B127 Tubulin Tails are Intrinsically Disordered Polyanions that Regulate Binding to other Proteins by Sequence as Well as Charge Dan L. Sackett. NICHD / NIH, Bethesda, MD, USA. Microtubules (MT) are built by polymerization of the alpha/beta tubulin heterodimer. The two tubulins are very similar to each other and are composed of a globular body containing about 95% of the protein mass and an unstructured C-terminal tail (CTT) that contains about 40% of the protein’s net negative charge. The CTT is the locus of most sequence differences between tubulin isotypes and also the target of most posttranslational modifications (PTMs). The tails extend from the tubulin body and from the outside surface of MT, and are often the first part of tubulin / MT that other proteins encounter. The high charge density on the tails can promote or oppose interaction interactions with other proteins. Surprisingly, perhaps, interactions with some proteins requires the negative charges, but are still highly modulated by sequence differences between tails from different isotypes. PTMs can add an additional, significant modulation of binding that can be determinative. Specific examples of each of these will be presented and discussed in the context of: Intrinsically Disordered Tails are Not Just Charged Strings. 1984-Pos Board B128 F Actin Bundling Dynamics and Stiffness of the TRIOBP-4 F Actin Bundle Justin J. Raupp, Laura K. Gunther, Yuwen Mei, Alexander Pattyn, Takeshi Sakamoto. Physics and Astronomy, Wayne State University, Detroit, MI, USA. The guanine nucleotide exchange factor (GEF) trio binding protein isoforms 4 and 5 (TRIOBP-4/-5) is an F-actin-bundling protein originally found to be associated with hearing loss both in humans and mice. The human and mouse TRIOBP protein is largely classified into three isoforms with a long isoform (TRIOBP-5) and two shorter isoforms equivalent to the N-terminus and C-terminus of TRIOBP-5, which are named TRIOBP-4 and TRIOBP-1 (or Tara), respectively. There are no common sequences between TRIOBP-1 and -4. TRIOBP-1 is ubiquitously expressed and has been shown to regulate adherens junctions and actin cytoskeleton reorganization mostly in stress fibers and cortical F-actin. In contrast, TRIOBP-4 and -5 are predominantly expressed in the inner ear and the retina of normal adult tissues, where they are believed to provide appropriate durability and rigidity for stereocilia of hair cells for normal hearing. Interestingly, different from fascin, which is intercalated between actin filaments via its two major actin binding sites, TRIOBP-4 and -5 form extremely dense F-actin bundles without detectable inter-filament spaces, raising the possibility that they more likely wrap around actin bundles. To date, the biological functions of TRIOBP-4/-5 have not been revealed in any other tissues except in inner ear hair cells for hearing. Recently, we identified actin binding sites of TRIOBP-4 isoforms and discovered that TRIOBP-4 expressed in cancer cells to facilitate cell migration speed. In this study, we will show F-actin bundling dynamics during actin polymerization under TRIF microscopy and determine the stiffness and durability of TRIOBP-4/F-actin bundles.
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reversal of the increase in oxygen consumption seen when INS832/13 cells, a rodent insulinoma cell line, are challenged with IAPP. In summary our studies suggest the possibility that humanin, and perhaps other mitochondriallyderived peptides, have chaperone-like properties that could reverse the pathological processes involved in the development of T2DM. 1982-Pos Board B126 Biophysics of Tardigrade Survival Samantha Piszkiewicz1, Aakash Mehta1, Thomas Boothby2, Bob Goldstein2,3, Gary Pielak1,4. 1 Chemistry, UNC Chapel Hill, Chapel Hill, NC, USA, 2Biology, UNC Chapel Hill, Chapel Hill, NC, USA, 3Lineberger Comprehensive Cancer Center, UNC Chapel Hill, Chapel Hill, NC, USA, 4Biochemistry and Biophysics, UNC Chapel Hill, Chapel Hill, NC, USA. Tardigrades, commonly known as water bears, are microorganisms that can survive extreme conditions: temperatures from 274 C to 151 C, pressures from 0 to 6,000 atmospheres, 1,000 times more radiation than the average animal, 10 days in space and over 20 years of dehydration. However, little is known about the mechanisms tardigrades use to survive these harsh environments. The Cytoplasmic Abundant Heat Soluble (CAHS) class of proteins is overexpressed by tardigrades under dehydration stress and the CAHS genes are essential for surviving dehydration. We have expressed these proteins in Escherichia coli. Circular dichroism spectropolarimetry and multidimensional, multinuclear NMR spectroscopy data indicate that the purified proteins are intrinsically disordered in dilute solution and form temperature-dependent reversible gel networks at higher concentrations. 1983-Pos Board B127 Tubulin Tails are Intrinsically Disordered Polyanions that Regulate Binding to other Proteins by Sequence as Well as Charge Dan L. Sackett. NICHD / NIH, Bethesda, MD, USA. Microtubules (MT) are built by polymerization of the alpha/beta tubulin heterodimer. The two tubulins are very similar to each other and are composed of a globular body containing about 95% of the protein mass and an unstructured C-terminal tail (CTT) that contains about 40% of the protein’s net negative charge. The CTT is the locus of most sequence differences between tubulin isotypes and also the target of most posttranslational modifications (PTMs). The tails extend from the tubulin body and from the outside surface of MT, and are often the first part of tubulin / MT that other proteins encounter. The high charge density on the tails can promote or oppose interaction interactions with other proteins. Surprisingly, perhaps, interactions with some proteins requires the negative charges, but are still highly modulated by sequence differences between tails from different isotypes. PTMs can add an additional, significant modulation of binding that can be determinative. Specific examples of each of these will be presented and discussed in the context of: Intrinsically Disordered Tails are Not Just Charged Strings. 1984-Pos Board B128 F Actin Bundling Dynamics and Stiffness of the TRIOBP-4 F Actin Bundle Justin J. Raupp, Laura K. Gunther, Yuwen Mei, Alexander Pattyn, Takeshi Sakamoto. Physics and Astronomy, Wayne State University, Detroit, MI, USA. The guanine nucleotide exchange factor (GEF) trio binding protein isoforms 4 and 5 (TRIOBP-4/-5) is an F-actin-bundling protein originally found to be associated with hearing loss both in humans and mice. The human and mouse TRIOBP protein is largely classified into three isoforms with a long isoform (TRIOBP-5) and two shorter isoforms equivalent to the N-terminus and C-terminus of TRIOBP-5, which are named TRIOBP-4 and TRIOBP-1 (or Tara), respectively. There are no common sequences between TRIOBP-1 and -4. TRIOBP-1 is ubiquitously expressed and has been shown to regulate adherens junctions and actin cytoskeleton reorganization mostly in stress fibers and cortical F-actin. In contrast, TRIOBP-4 and -5 are predominantly expressed in the inner ear and the retina of normal adult tissues, where they are believed to provide appropriate durability and rigidity for stereocilia of hair cells for normal hearing. Interestingly, different from fascin, which is intercalated between actin filaments via its two major actin binding sites, TRIOBP-4 and -5 form extremely dense F-actin bundles without detectable inter-filament spaces, raising the possibility that they more likely wrap around actin bundles. To date, the biological functions of TRIOBP-4/-5 have not been revealed in any other tissues except in inner ear hair cells for hearing. Recently, we identified actin binding sites of TRIOBP-4 isoforms and discovered that TRIOBP-4 expressed in cancer cells to facilitate cell migration speed. In this study, we will show F-actin bundling dynamics during actin polymerization under TRIF microscopy and determine the stiffness and durability of TRIOBP-4/F-actin bundles.