Biomedical subjects

Margaret Sunde

Publications and source records attributed to Margaret Sunde.

13 recordsLinked to original sources

PubMed2006

Identification of the key LMO2-binding determinants on Ldb1.

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS- and beta-casein.

Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate; the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.

Amyloid↗

PubMed2004

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex.

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity.

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.

Amyloid↗

PubMed2004

TC-1 is a novel tumorigenic and natively disordered protein associated with thyroid cancer.

A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.

Apoptosis↗

PubMed2004

Backbone dynamics of oxidised and reduced forms of human atrial natriuretic peptide.

The backbone dynamics of the 28 residue 15N-labelled human atrial natriuretic peptide have been examined by 15N NMR methods. 15N R1, R2 and [1H]-15N NOE values were determined for the oxidised and reduced forms of the peptide (ANPox and ANPrd, respectively), and analysed using reduced spectral density mapping and an extended model-free approach. The two forms possessed correlation times for overall molecular motion of 4.7 ns and were highly flexible, with substantial contributions to relaxation processes from internal motions on picosecond to nanosecond time scales. Reduction of the Cys7-Cys23 disulphide bond to form ANPrd produced a very dynamic linear peptide with a mean overall order parameter of 0.2; the intramolecular cross-link in ANPox increased this to a mean value of 0.4. A simple model for segmental backbone motion accounted for the R2 values of both species using only two variable parameters, indicating that relaxation is dominated by interactions with sites <7 residues distant in the covalent network and that changes in the conformation of the disulphide bond lead to significant chemical exchange broadening in ANPox. The contributions of backbone dynamics to configurational entropy were determined and accounted for the different receptor binding affinities of cyclised and linear natriuretic peptides.

Atrial Natriuretic Factor↗

PubMed2004

The power of two: protein dimerization in biology.

The self-association of proteins to form dimers and higher-order oligomers is a very common phenomenon. Recent structural and biophysical studies show that protein dimerization or oligomerization is a key factor in the regulation of proteins such as enzymes, ion channels, receptors and transcription factors. In addition, self-association can help to minimize genome size, while maintaining the advantages of modular complex formation. Oligomerization, however, can also have deleterious consequences when nonnative oligomers associated with pathogenic states are generated. Specific protein dimerization is integral to biological function, structure and control, and must be under substantial selection pressure to be maintained with such frequency throughout biology.

Allosteric Regulation↗

PubMed2003

The C-terminal domain of Eos forms a high order complex in solution.

Ikaros family transcription factors play important roles in the control of hematopoiesis. Family members are predicted to contain up to six classic zinc fingers that are arranged into N- and C-terminal domains. The N-terminal domain is responsible for site-specific DNA binding, whereas the C-terminal domain primarily mediates the homo- and hetero-oligomerization between family members. Although the mechanisms of action of these proteins are not completely understood, the zinc finger domains are known to play a central role. In the current study, we have sought to understand the physical and functional properties of these domains, in particular the C-terminal domain. We show that the N-terminal domain from Eos, and not its C-terminal region, is required to recognize GGGA consensus sequences. Surprisingly, in contrast to the behavior exhibited by Ikaros, the C-terminal domain of Eos inhibits the DNA-binding activity of the full-length protein. In addition, we have used a range of biophysical techniques to demonstrate that the C-terminal domain of Eos mediates the formation of complexes that consist of nine or ten molecules. These results constitute the first direct demonstration that Ikaros family proteins can form higher order complexes in solution, and we discuss this unexpected result in the context of what is currently known about the family members and their possible mechanism of action.

Amino Acid Sequence↗

PubMed2003

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation.

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.

Amino Acid Sequence↗

PubMed2003

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation.

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

Amyloid↗

PubMed2002

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I.

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.

Amino Acid Sequence↗

PubMed2002

Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme.

Hydrogen exchange experiments monitored by NMR and mass spectrometry reveal that the amyloidogenic D67H mutation in human lysozyme significantly reduces the stability of the beta-domain and the adjacent C-helix in the native structure. In addition, mass spectrometric data reveal that transient unfolding of these regions occurs with a high degree of cooperativity. This behavior results in the occasional population of a partially structured intermediate in which the three alpha-helices that form the core of the alpha-domain still have native-like structure, whereas the beta-domain and C-helix are simultaneously substantially unfolded. This finding suggests that the extensive intermolecular interactions that will be possible in such a species are likely to initiate the aggregation events that ultimately lead to the formation of the well-defined fibrillar structures observed in the tissues of patients carrying this mutation in the lysozyme gene.

Genetic Variation↗

PubMed2002

Zinc fingers--folds for many occasions.

Zinc finger domains (ZnFs) are common, relatively small protein motifs that fold around one or more zinc ions. In addition to their role as a DNA-binding module, ZnFs have recently been shown to mediate protein:protein and protein:lipid interactions. This small zinc-ligating domain, often found in clusters containing fingers with different binding specificities, can facilitate multiple, often independent intermolecular interactions between nucleic acids and proteins. Classical ZnFs, typified by TFIIIA, ligate zinc via pairs of cysteine and histidine residues but there are at least 14 different classes of Zn fingers, which differ in the nature and arrangement of their zinc-binding residues. Some GATA-type ZnFs can bind to both DNA and a variety of other proteins. Thus proteins with multiple GATA-type fingers can play a complex role in regulating transcription through the interplay of these different binding selectivities and affinities. Other ZnFs have more specific functions, such as DNA-binding ZnFs in the nuclear hormone receptor proteins and small-molecule-binding ZnFs in protein kinase C. Some classes of ZnFs appear to act exclusively in protein-only interactions. These include the RING family of ZnFs that are involved in ubiquitination processes and in the assembly of large protein complexes, LIM, TAZ, and PHD domains. We review the similarities and differences in structure and functions of different ZnF classes and highlight the versatility of this fold.

Amino Acid Motifs↗