Mutations in the Vicinity of the IRAK3 Guanylate Cyclase Center Impact Its Subcellular Localization and Ability to Modulate Inflammatory Signaling in Immortalized Cell Lines.

Interleukin-1 receptor-associated kinase 3 (IRAK3) modulates the magnitude of cellular responses to ligands perceived by interleukin-1 receptors (IL-1Rs) and Toll-like receptors (TLRs), leading to decreases in pro-inflammatory cytokines and suppressed inflammation. The molecular mechanism of IRAK3's action remains unknown. IRAK3 functions as a guanylate cyclase, and its cGMP product suppresses lipopolysaccharide (LPS)-induced nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) activity. To understand the implications of this phenomenon, we expanded the structure-function analyses of IRAK3 through site-directed mutagenesis of amino acids known or predicted to impact different activities of IRAK3. We verified the capacity of the mutated IRAK3 variants to generate cGMP in vitro and revealed residues in and in the vicinity of its GC catalytic center that impact the LPS-induced NFκB activity in immortalized cell lines in the absence or presence of an exogenous membrane-permeable cGMP analog. Mutant IRAK3 variants with reduced cGMP generating capacity and differential regulation of NFκB activity influence subcellular localization of IRAK3 in HEK293T cells and fail to rescue IRAK3 function in IRAK3 knock-out THP-1 monocytes stimulated with LPS unless the cGMP analog is present. Together, our results shed new light on the mechanism by which IRAK3 and its enzymatic product control the downstream signaling, affecting inflammatory responses in immortalized cell lines.

IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

DCC mutation update: Congenital mirror movements, isolated agenesis of the corpus callosum, and developmental split brain syndrome.

The deleted in colorectal cancer (DCC) gene encodes the netrin-1 (NTN1) receptor DCC, a transmembrane protein required for the guidance of commissural axons. Germline DCC mutations disrupt the development of predominantly commissural tracts in the central nervous system (CNS) and cause a spectrum of neurological disorders. Monoallelic, missense, and predicted loss-of-function DCC mutations cause congenital mirror movements, isolated agenesis of the corpus callosum (ACC), or both. Biallelic, predicted loss-of-function DCC mutations cause developmental split brain syndrome (DSBS). Although the underlying molecular mechanisms leading to disease remain poorly understood, they are thought to stem from reduced or perturbed NTN1 signaling. Here, we review the 26 reported DCC mutations associated with abnormal CNS development in humans, including 14 missense and 12 predicted loss-of-function mutations, and discuss their associated clinical characteristics and diagnostic features. We provide an update on the observed genotype-phenotype relationships of congenital mirror movements, isolated ACC and DSBS, and correlate this to our current understanding of the biological function of DCC in the development of the CNS. All mutations and their associated phenotypes were deposited into a locus-specific LOVD (https://databases.lovd.nl/shared/genes/DCC).

Functional Characterization of the Unique Terminal Thioesterase Domain from Polymyxin Synthetase.

Polymyxins remain one of the few antibiotics available for treating antibiotic resistant bacteria. Here we describe polymyxin B thioesterase which performs the final step in polymyxin B biosynthesis. Isolated thioesterase catalyzed cyclization of an N-acetylcystamine polymyxin B analogue to form polymyxin B. The thioesterase contained a catalytic cysteine unlike most thioesterases which possess a serine. Supporting this, incubation of polymyxin B thioesterase with reducing agents abolished enzymatic activity, while mutation of the catalytic cysteine to serine significantly decreased activity. NMR spectroscopy demonstrated that uncyclized polymyxin B was disordered in solution, unlike other thioesterase substrates which adopt a transient structure similar to their product. Modeling showed the thioesterase substrate-binding cleft was highly negatively charged, suggesting a mechanism for the cyclization of the substrate. These studies provide new insights into the role of polymyxin thioesterase in polymyxin biosynthesis and highlight its potential use for the chemoenzymatic synthesis of polymyxin lipopeptides.

Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders.

Compound heterozygous FXN mutations and clinical outcome in friedreich ataxia.

OBJECTIVE: Friedreich ataxia (FRDA) is an inherited neurodegenerative disease characterized by ataxia and cardiomyopathy. Homozygous GAA trinucleotide repeat expansions in the first intron of FXN occur in 96% of affected individuals and reduce frataxin expression. Remaining individuals are compound heterozygous for a GAA expansion and a FXN point/insertion/deletion mutation. We examined disease-causing mutations and the impact on frataxin structure/function and clinical outcome in FRDA. METHODS: We compared clinical information from 111 compound heterozygotes and 131 individuals with homozygous expansions. Frataxin mutations were examined using structural modeling, stability analyses and systematic literature review, and categorized into four groups: (1) homozygous expansions, and three compound heterozygote groups; (2) null (no frataxin produced); (3) moderate/strong impact; and (4) minimal impact. Mean age of onset and the presence of cardiomyopathy and diabetes mellitus were compared using regression analyses. RESULTS: Mutations in the hydrophobic core of frataxin affected stability whereas surface residue mutations affected interactions with iron sulfur cluster assembly and heme biosynthetic proteins. The null group of compound heterozygotes had significantly earlier age of onset and increased diabetes mellitus, compared to the homozygous expansion group. There were no significant differences in mean age of onset between homozygotes and the minimal and moderate/strong impact groups. INTERPRETATION: In compound heterozygotes, expression of partially functional mutant frataxin delays age of onset and reduces diabetes mellitus, compared to those with no frataxin expression from the non-expanded allele. This integrated analysis of categorized frataxin mutations and their correlation with clinical outcome provide a definitive resource for investigating disease pathogenesis in FRDA.

Characterization of the Polymyxin D Synthetase Biosynthetic Cluster and Product Profile of Paenibacillus polymyxa ATCC 10401.

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-α,γ-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D1 and D2 but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D1 and D2 exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser3 polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.

Domain structure and function of matrix metalloprotease 23 (MMP23): role in potassium channel trafficking.

MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein-protein and protein-lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.

Intracellular trafficking of the KV1.3 potassium channel is regulated by the prodomain of a matrix metalloprotease.

Matrix metalloproteases (MMPs) are endopeptidases that regulate diverse biological processes. Synthesized as zymogens, MMPs become active after removal of their prodomains. Much is known about the metalloprotease activity of these enzymes, but noncanonical functions are poorly defined, and functions of the prodomains have been largely ignored. Here we report a novel metalloprotease-independent, channel-modulating function for the prodomain of MMP23 (MMP23-PD). Whole-cell patch clamping and confocal microscopy, coupled with deletion analysis, demonstrate that MMP23-PD suppresses the voltage-gated potassium channel KV1.3, but not the closely related KV1.2 channel, by trapping the channel intracellularly. Studies with KV1.2-1.3 chimeras suggest that MMP23-PD requires the presence of the KV1.3 region from the S5 trans-membrane segment to the C terminus to modulate KV1.3 channel function. NMR studies of MMP23-PD reveal a single, kinked trans-membrane α-helix, joined by a short linker to a juxtamembrane α-helix, which is associated with the surface of the membrane and protected from exchange with the solvent. The topological similarity of MMP23-PD to KCNE1, KCNE2, and KCNE4 proteins that trap KV1.3, KV1.4, KV3.3, and KV3.4 channels early in the secretory pathway suggests a shared mechanism of channel regulation. MMP23 and KV1.3 expression is enhanced and overlapping in colorectal cancers where the interaction of the two proteins could affect cell function.

A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily.

Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.

Intrinsic protein flexibility in regulation of cell proliferation: advantages for signaling and opportunities for novel therapeutics.

It is now widely recognized that intrinsically disordered (or unstructured) proteins (IDPs, or IUPs) are found in organisms from all kingdoms of life. In eukaryotes, IDPs are highly abundant and perform a wide range of biological functions, including regulation and signaling. Despite increased interest in understanding the structural biology of IDPs, questions remain regarding the mechanisms through which disordered proteins perform their biological function(s). In other words, what are the relationships between disorder and function for IDPs? Several excellent reviews have recently been published that discuss the structural properties of IDPs.1-3 Here, we discuss two IDP systems which illustrate features of dynamic complexes. In the first section, we discuss two IDPs, p21 and p27, which regulate the mammalian cell division cycle by inhibiting cyclin-dependent kinases (Cdks). In the second section, we discuss recent results from Follis, Hammoudeh, Metallo and coworkers demonstrating that the IDP Myc can be bound and inhibited by small molecules through formation of dynamic complexes. Previous studies have shown that polypeptide segments of p21 and p27 are partially folded in isolation and fold further upon binding their biological targets. Interestingly, some portions of p27 which bind to and inhibit Cdk2/cyclin A remain flexible in the bound complex. This residual flexibility allows otherwise buried tyrosine residues within p27 to be phosphorylated by nonreceptor tyrosine kinases (NRTKs). Tyrosine phosphorylation relieves kinase inhibition, triggering Cdk2-mediated phosphorylation of a threonine residue within the flexible C-terminus of p27. This, in turn, marks p27 for ubiquitination and proteasomal degradation, unleashing full Cdk2 activity which drives cell cycle progression. p27, thus, constitutes a conduit for transmission of proliferative signals via posttranslational modifications. Importantly, activation of the p27 signaling conduit by oncogenic NRTKs contributes to tumorigenesis in some human cancers, including chronic myelogenous leukemia (CML)9 and breast cancer.10 Another IDP with important roles in human cancer is the proto-oncoprotein, Myc. Myc is a DNA binding transcription factor which critically drives cell proliferation in many cell types and is often deregulated in cancer. Myc is intrinsically disordered in isolation and folds upon binding another IDP, Max and DNA. Follis, Hammoudeh, Metallo and coworkers identified small molecules which bind disordered regions of Myc and inhibit its heterodimerization with Max. Importantly, these small molecules- through formation of dynamic complexes with Myc-have been shown to inhibit Myc function in vitro and in cellular assays, opening the door to IDP-targeted therapeutics in the future. The p21/p27 and Myc systems illustrate, from different perspectives, the role of dynamics in IDP function. Dynamic fluctuations are critical for p21/p27 signaling while the dynamic free state of Myc may represent a therapeutically approachable anticancer target. Herein we review the current state of knowledge related to these two topics in IDP research.

A novel deletion-insertion mutation identified in exon 3 of FXN in two siblings with a severe Friedreich ataxia phenotype.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease most commonly caused by a GAA trinucleotide repeat expansion in the first intron of FXN, which reduces expression of the mitochondrial protein frataxin. Approximately 98% of individuals with FRDA are homozygous for GAA expansions, with the remaining 2% compound heterozygotes for a GAA expansion and a point mutation within FXN. Two siblings with early onset of symptoms experienced rapid loss of ambulation by 8 and 10 years. Diagnostic testing for FRDA demonstrated one GAA repeat expansion of 1010 repeats and one non-expanded allele. Sequencing all five exons of FXN identified a novel deletion-insertion mutation in exon 3 (c.371_376del6ins15), which results in a modified frataxin protein sequence at amino acid positions 124-127. Specifically, the amino acid sequence changes from DVSF to VHLEDT, increasing frataxin from 211 residues to 214. Using the known structure of human frataxin, a theoretical 3D model of the mutant protein was developed. In the event that the modified protein is expressed and stable, it is predicted that the acidic interface of frataxin, known to be involved in iron binding and interactions with the iron-sulphur cluster assembly factor IscU, would be impaired.

Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits.

It is now widely recognized that intrinsically unstructured (or disordered) proteins (IUPs or IDPs) are found in organisms from all kingdoms of life. In eukaryotes, IUPs are highly abundant and perform a wide range of biological functions, including regulation and signaling. Despite an increased level of interest in understanding the structural biology of IUPs and IDPs, questions regarding the mechanisms through which disordered proteins perform their biological function(s) remain. In other words, what are the relationships between disorder and function for IUPs? There are several excellent reviews that discuss the structural properties of IUPs and IDPs since 2005 [Receveur-Brechot, V., et al. (2006) Proteins 62, 24-45; Mittag, T., and Forman-Kay, J. D. (2007) Curr. Opin. Struct. Biol. 17, 3-14; Dyson, H. J., and Wright, P. E. (2005) Nat. Rev. Mol. Cell Biol. 6, 197-208]. Here, we briefly review general concepts pertaining to IUPs and then discuss our structural, biophysical, and biochemical studies of two IUPs, p21 and p27, which regulate the mammalian cell division cycle by inhibiting cyclin-dependent kinases (Cdks). Some segments of these two proteins are partially folded in isolation, and they fold further upon binding their biological targets. Interestingly, some portions of p27 remain flexible after binding to and inhibiting the Cdk2-cyclin A complex. This residual flexibility allows otherwise buried tyrosine residues within p27 to be phosphorylated by non-receptor tyrosine kinases (NRTKs). Tyrosine phosphorylation relieves kinase inhibition, triggering Cdk2-mediated phosphorylation of a threonine residue within the flexible C-terminus of p27. This, in turn, marks p27 for ubiquitination and proteasomal degradation, unleashing full Cdk2 activity which drives cell cycle progression. p27, thus, constitutes a conduit for transmission of proliferative signals via post-translational modifications. The term "conduit" is used here to connote a means of transmission of molecular signals which, in the case of p27, correspond to tyrosine and threonine phosphorylation, ubiquitination, and, ultimately, proteolytic degradation. Transmission of these multiple signals is enabled by the inherent flexibility of p27 which persists even after tight binding to the Cdk2-cyclin A complex. Importantly, activation of the p27 signaling conduit by oncogenic NRTKs contributes to tumorigenesis in some human cancers, including chronic myelogenous leukemia (CML) [Grimmler, M., et al. (2007) Cell 128, 269-280] and breast cancer [Chu, I., et al. (2007) Cell 128, 281-294]. Other IUPs may participate in conceptually similar molecular signaling conduits, and dysregulation of these putative conduits may contribute to other human diseases. Detailed study of these IUPs, both alone and within functional complexes, is required to test these hypotheses and to more fully understand the relationships between protein disorder and biological function.

Thermodynamic characterization of interactions between p27(Kip1) and activated and non-activated Cdk2: intrinsically unstructured proteins as thermodynamic tethers.

The cyclin-dependent kinase inhibitor (CKI) p27Kip1 plays a critical role in cell cycle regulation by binding and inhibiting (or activating) various cyclin-dependent kinase (Cdk)/cyclin complexes. Thermal denaturation monitored by circular dichroism (CD) and isothermal titration calorimetry (ITC) were used to determine the relative stabilities and affinities of p27-KID (p27 kinase inhibitory domain) complexes with activated Cdk2 (phosphorylated at Thr160; P-Cdk2) and non-activated forms of Cdk2 and/or cyclin A. Phosphorylation of residue Thr160 only slightly increases the thermal stability of Cdk2, and its binary complexes with cyclin A and p27-KID. The p27-KID/P-Cdk2/cyclin A or p27-KID/Cdk2/cyclin A ternary complexes exhibited significantly higher thermal stabilities compared to the binary complexes (P-Cdk2/cyclin A or Cdk2/cyclin A). Differences in T(m) values between the binary and ternary complexes with P-Cdk2 and Cdk2 were +25.9 and +20.4 degrees C, respectively. These results indicate that the ternary complex with phosphorylated Cdk2 is stabilized to a larger extent than the non-phosphorylated complex. The free energy of association (deltaG(A)) for formation of the two ternary complexes was more favorable than for the binary complexes, indicating that a significantly smaller population of free components existed when all three components were present. These data indicate that p27-KID, which is intrinsically disordered in solution, acts as a thermodynamic tether when bound within the ternary complexes. It is proposed that thermodynamic tethering may be a general phenomena associated with intrinsically unstructured proteins (IUPs) which often function by binding to multiple partners in multi-protein assemblies.

Expression and Purification of a Matrix Metalloprotease Transmembrane Domain in Escherichia coli.

Membrane tethered matrix metalloproteases are bound to the plasma membrane by a glycosylphosphatidylinositol-anchor or a transmembrane domain. To date, most studies of membrane-bound matrix metalloprotease have focused on the globular catalytic and protein-protein interaction domains of these enzymes. However, the transmembrane domains have been poorly studied even though they are known to mediate intracellular signaling via interaction with various cellular proteins. The expression and purification of the transmembrane domain of these proteins can be challenging due to their hydrophobic nature. In this chapter we describe the purification of a transmembrane domain for a membrane-bound matrix metalloprotease expressed in E. coli and its initial characterization by NMR spectroscopy.

Mutations in DCC cause isolated agenesis of the corpus callosum with incomplete penetrance.

Brain malformations involving the corpus callosum are common in children with developmental disabilities. We identified DCC mutations in four families and five sporadic individuals with isolated agenesis of the corpus callosum (ACC) without intellectual disability. DCC mutations result in variable dominant phenotypes with decreased penetrance, including mirror movements and ACC associated with a favorable developmental prognosis. Possible phenotypic modifiers include the type and location of mutation and the sex of the individual.

Neuroendocrine manifestations of phospholipid antibody disease identified by long-term follow-up study of patients with phospholipid antibodies.

UNLABELLED: Recurrent clinical thrombotic episodes and/or recurrent fetal wastage are the clinical features of phospholipid antibody (aPL) syndrome, which is characterized by a bland thrombosis, but is not inflammatory, as is found in other connective tissue diseases such as systemic lupus erythematosus (SLE). Previous reports have suggested that some patients with primary aPL syndrome may progress to develop other autoimmune diseases, including inflammatory diseases such as SLE. The aim of this study was to determine the long-term outcome of women with aPL antibodies, with regard to progression of their underlying autoimmune disease. To that end, a retrospective study was made of women with aPL and primary aPL syndromes who had been followed at our institution for a minimum of 3 years. Charts were reviewed, patients interviewed, and laboratory tests were performed to determine whether the clinical nature of the disease and/or its autoantibody profile had changed. Thirty patients were enrolled into the study (29 with aPL syndrome, 1 with consistent aPL and no syndrome). Follow-up ranged from 3 to 22 years. Results were as follows: The autoimmune clinical features were unchanged in 27 patients, but 3 patients developed inflammatory disease, presenting with nasal chondritis (2), cutaneous vasculitis (3), and mucosal ulcer (1). In each case, these changes occurred during pregnancy or the immediate postpartum period. One patient fulfilled criteria for SLE as seen by a change in her autoantibody profile. Another incidental finding was that three other patients were diagnosed with papillary thyroid cancer, two being diagnosed during the follow-up period. IN CONCLUSION: (1) Inflammatory disease may develop in some patients with aPL and appears to be set off by pregnancy, a known trigger for clinical thrombotic events in aPL patients. (2) Thyroid cancer may be associated with aPL, and this association warrants further study with larger number of patients.

Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils.

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337-Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37 degrees C, a p53 tetramerization domain mutant (p53tet-R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild-type (p53tet-wt) did not. We characterized these proteins by equilibrium denaturation, 13C(alpha) secondary chemical shifts, (1H)-15N heteronuclear NOEs, and H/D exchange. Although p53tet-R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pK(a) values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet-R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet-R337H stability resulted in an increased propensity to form amyloid fibrils.

Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant.

The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amyloid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thioflavin T showed that p53tet-wt and p53tet-R337H adopt an alternative beta-sheet conformation (p53tet-wt-beta and p53tet-R337H-beta, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elevated temperatures. Electron micrographs showed that the alternative conformations for p53tet-wt (p53tet-wt-beta) and p53tet-R337H (p53tet-R337H-beta) were supramolecular structures best described as "molecular ribbons". FT-IR analysis demonstrated that the mechanism of amyloid-like fibril formation involved unfolding of the p53tet-wt beta-strands, followed by unfolding of the alpha-helices, followed finally by formation of beta-strand-containing structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 degrees C) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 degrees C over 20 hours. Interestingly, unlike other amyloid-forming proteins, p53tet-wt-beta and p53tet-R337H-beta disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassembly at pH 8.5 was independent of both. Finally, we propose that the significantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.

Beyond loss of frataxin: the complex molecular pathology of Friedreich ataxia.

Friedreich ataxia (FRDA) is a devastating neurodegenerative disease caused by mutations in the frataxin gene (FXN). Frataxin is an essential protein which localizes to the mitochondria and is required for the synthesis of iron-sulfur clusters and heme. Most individuals with FRDA are homozygous for trinucleotide GAA.TTC repeat expansions in intron 1 of FXN. The instability of these GAA.TTC repeats, the formation of non-B DNA GAA.TTC structures, and accompanying epigenetic changes lead to reduced FXN transcript and frataxin protein. This 'loss of frataxin' is considered the main driver of disease pathology with mitochondria-rich tissues such as the heart and the brain most affected. While our understanding of FRDA etiology has advanced in recent years, exactly how reduced frataxin leads to disease remains largely unknown. Most therapeutic strategies aim to increase frataxin, yet there are other underlying aspects of the molecular pathology that could impact disease progression and severity. These include RNA toxicity due to antisense RNAs, dysregulated splicing and microRNAs, and repeat-associated protein toxicity via RAN translation. Here we review the diverse array of molecular events that have been shown to influence clinical outcome in FRDA. We also examine additional pathogenic factors from other trinucleotide repeat diseases which could be potentially important in FRDA.