Summary Thyroglobulin is the protein precursor of thyroid hormones, which are essential for growth, development and control of metabolism in vertebrates 1 , 2 . Hormone synthesis from thyroglobulin (TG) occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis 3 . Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified and the lack of a three-dimensional structure of TG has prevented mechanistic understanding 4 . Here we present the structure of full-length human thyroglobulin at ~3.5 Å resolution determined by electron cryomicroscopy (cryo-EM). We identified all hormonogenic tyrosine pairs in the structure and verified them via site-directed mutagenesis and in vitro hormone production assays using human TG expressed in HEK cells. Analysis revealed that proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. Transferring the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose binding protein (MBP) yielded hormone production with efficiency comparable to TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.
To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid--(1-40) peptide (A). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to A is oligomer specific. The dimers and tetramers of stefin B, which bind A, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit A fibril formation. When expressed in cultured cells, stefin B co-localizes with A intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the A epitope. Thus, stefin B is another APP/A-binding protein in vitro and likely in cells.Neurodegenerative diseases present a huge burden in the developed world's aging population. They are all in one way or another connected to aberrant protein folding and aggregation of the proteins involved (1). Various protein conformational disorders of the central and peripheral nervous system are known, which often appear sporadically but also run in families. These are among others: Parkinson and Alzheimer diseases, dementia with Lewy bodies, vascular and fronto-temporal dementia, and amyotrophic lateral sclerosis.The A peptide implicated in Alzheimer disease pathology is a cleavage product of the membrane A precursor protein (APP).3 It is the main constituent of extracellular amyloid plaques, however, together with its oligomers, it also resides intracellularly (2). It has been shown that A oligomers prepared in vitro and those extracted from living cells exert cytotoxicity and cause symptoms of reversible memory loss in animal models (3). Amyloid protein oligomers have special structural properties, which are reflected in a common antioligomer antibody (4). This antibody not only binds the oligomers against which it was raised but also binds chaperones and some other proteins involved in disaggregating protein aggregates in cells (5). A-binding proteins, the so called "amateur chaperones," were suggested to have a potential in Alzheimer disease therapy (6, 7).It has been shown before that human cystatin C is an A-binding protein (8). Cystatins are single chain proteins that inhibit cysteine cathepsins (9). Human stefin B (also known as cystatin B) is a member of subfamily A of cystatins, classified as family I25 in the MEROPS scheme (10). Stefin B, a protein of 98 amino acid residues and 1 Cys, is predominantly intracellular, whereas cystatin C, a protein of 120 residues and 2 disulfide bonds, is a secretory protein. Three-dimensional structures of stefins and cystatin C have been determined, among others, the solution structure of stefin A (11) and cystatin C (12, 13).Human cystatin C has been found as a constituent of senile plaques of Alzheimer disease patients (14) and stefins A and B have also been reported to localize to a...
Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.
Serum paraoxonase-1 (PON1) is the most studied member of the group of paraoxonases (PONs). This enzyme possesses three enzymatic activities: lactonase, arylesterase, and paraoxonase activity. PON1 and its isoforms play an important role in drug metabolism as well as in the prevention of cardiovascular and neurodegenerative diseases. Although all three members of the PON family have the same origin and very similar amino acid sequences, they have different functions and are found in different locations. PONs exhibit substrate promiscuity, and their true physiological substrates are still not known. However, possible substrates include homocysteine thiolactone, an analogue of natural quorum-sensing molecules, and the recently discovered derivatives of arachidonic acid—bioactive δ-lactones. Directed evolution, site-directed mutagenesis, and kinetic studies provide comprehensive insights into the active site and catalytic mechanism of PON1. However, there is still a whole world of mystery waiting to be discovered, which would elucidate the substrate promiscuity of a group of enzymes that are so similar in their evolution and sequence yet so distinct in their function.
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