Publications by authors named "Laura A Sikkink"

11 Publications

  • Page 1 of 1

Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

Biochemistry 2013 Feb 6;52(7):1249-59. Epub 2013 Feb 6.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN 55905, USA.

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.
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http://dx.doi.org/10.1021/bi301500dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587134PMC
February 2013

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain.

Biochem Biophys Res Commun 2011 Dec 19;416(3-4):367-71. Epub 2011 Nov 19.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
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http://dx.doi.org/10.1016/j.bbrc.2011.11.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242870PMC
December 2011

Light chain amyloidosis - current findings and future prospects.

Curr Protein Pept Sci 2009 Oct;10(5):500-508

Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.

Systemic light chain amyloidosis (AL) is one of several protein misfolding diseases and is characterized by extracellular deposition of immunoglobulin light chains in the form of amyloid fibrils [1]. Immunoglobulin (Ig) proteins consist of two light chains (LCs) and two heavy chains (HCs) that ordinarily form a heterotetramer which is secreted by a plasma cell. In AL, however, a monoclonal plasma cell population produces an abundance of a pathogenic LC protein. In this case, not all of the LCs pair with the HCs, and free LCs are secreted into circulation. The LC-HC dimer is very stable, and losing this interaction may result in an unstable LC protein [2]. Additionally, somatic mutations are thought to cause amyloidogenic proteins to be less stable compared to non-amyloidogenic proteins [3-5], leading to protein misfolding and amyloid fibril formation. The amyloid fibrils cause tissue damage and cell death, leading to patient death within 12-18 months if left untreated [6]. Current therapies are harsh and not curative, including chemotherapy and autologous stem cell transplants. Studies of protein pathogenesis and fibril formation mechanisms may lead to better therapies with an improved outlook for patient survival. Much has been done to determine the molecular factors that make a particular LC protein amyloidogenic and to elucidate the mechanism of amyloid fibril formation. Anthony Fink's work, particularly with discerning the role of intermediates in the fibril formation pathway, has made a remarkable impact in the field of amyloidosis research. This review provides a general overview of the current state of AL research and also attempts to capture the most recent ideas and knowledge generated from the Fink laboratory.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898330PMC
http://dx.doi.org/10.2174/138920309789351949DOI Listing
October 2009

Structural and functional studies of truncated hemolysin A from Proteus mirabilis.

J Biol Chem 2009 Aug 3;284(33):22297-22309. Epub 2009 Jun 3.

Physiology and Biomedical Imaging, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905.

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel beta-helix with three adjoining segments of anti-parallel beta-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete beta-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel beta-sheet interactions between neighboring beta-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, beta-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.
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http://dx.doi.org/10.1074/jbc.M109.014431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755953PMC
August 2009

Mutations in specific structural regions of immunoglobulin light chains are associated with free light chain levels in patients with AL amyloidosis.

PLoS One 2009 13;4(4):e5169. Epub 2009 Apr 13.

Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

Background: The amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL), each patient has a unique monoclonal immunoglobulin light chain (LC) that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains.

Methodology/principal Findings: We performed sequence alignments on the variable domain of 50 kappa and 91 lambda AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition.

Conclusions: Our results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005169PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664898PMC
October 2009

Biologic and genetic characterization of the novel amyloidogenic lambda light chain-secreting human cell lines, ALMC-1 and ALMC-2.

Blood 2008 Sep 20;112(5):1931-41. Epub 2008 Jun 20.

Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA.

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge, no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach, we established the genetic relationship between the cell lines and the primary patient cells, and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly, we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity, the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover, these cell lines will provide an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology.
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http://dx.doi.org/10.1182/blood-2008-03-143040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2518895PMC
September 2008

Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain.

Biophys Chem 2008 Jun 18;135(1-3):25-31. Epub 2008 Mar 18.

Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO(4)(2-) conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.
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http://dx.doi.org/10.1016/j.bpc.2008.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441971PMC
June 2008

Biochemical and aggregation analysis of Bence Jones proteins from different light chain diseases.

Amyloid 2008 Mar;15(1):29-39

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

Deposition of immunoglobulin light chains is a result of clonal proliferation of monoclonal plasma cells that secrete free immunoglobulin light chains, also called Bence Jones proteins (BJP). These BJP are present in circulation in large amounts and excreted in urine in various light chain diseases such as light chain amyloidosis (AL), light chain deposition disease (LCDD) and multiple myeloma (MM). BJP from patients with AL, LCDD and MM were purified from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results show that LCDD and MM proteins have the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJP at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. The aggregates formed under in vitro conditions suggested that the various proteins derived from patients with different light chain diseases might follow different aggregation pathways.
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http://dx.doi.org/10.1080/13506120701815324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556190PMC
March 2008

The effects of sodium sulfate, glycosaminoglycans, and Congo red on the structure, stability, and amyloid formation of an immunoglobulin light-chain protein.

Protein Sci 2006 Jul 2;15(7):1710-22. Epub 2006 Jun 2.

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

Light-chain amyloidosis (AL) is characterized by immunoglobulin light-chain fragments aggregating into amyloid fibrils that deposit extracellularly in vital organs such as the kidney, the heart, and the liver, resulting in tissue degeneration and organ failure, leading to death. Cardiac involvement is found in 50% of AL patients and presents the most severe cases with a life expectancy of less than a year after diagnosis. In this study, we have characterized the variable domain of a cardiac AL patient light chain called AL-09. AL-09 folds as a beta-sheet and is capable of forming amyloid fibrils both in the presence of sodium sulfate and in self-seeded reactions under physiological conditions. Glycosaminoglycans such as dermatan sulfate and heparin promote amyloid formation of self-seeded AL-09 reactions, while the glycosaminoglycan chondroitin sulfate A stabilized oligomeric intermediates and did not elongate the preformed fibrils (nucleus) present in the reaction. Finally, the histological dye Congo red, known to bind to the cross beta-sheet structure of amyloid fibrils, inhibits AL-09 amyloid fibril formation in the presence of sodium sulfate and in self-seeded reactions. This paper provides insight into the impact of different reagents on light-chain stability, structure, amyloid fibril formation, and inhibition.
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http://dx.doi.org/10.1110/ps.051997606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242560PMC
July 2006

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure.

Protein Sci 2006 Mar 1;15(3):522-32. Epub 2006 Feb 1.

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Scottsdale, AZ 85259, USA.

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.
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http://dx.doi.org/10.1110/ps.051810106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2249773PMC
March 2006

Human thiopurine S-methyltransferase pharmacogenetics: variant allozyme misfolding and aggresome formation.

Proc Natl Acad Sci U S A 2005 Jun 20;102(26):9394-9. Epub 2005 Jun 20.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein. In this study, we tested the hypothesis that TPMT*3A might result in protein misfolding and aggregation. We observed that TPMT*3A forms aggresomes in cultured cells and that it aggregates in vitro, functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT*3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs.
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http://dx.doi.org/10.1073/pnas.0502352102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1153717PMC
June 2005