Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml1p mutants revealed that the N-terminal 8−20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.
SummaryThis chapter presents a broad and a historical overview of the problem of protein structure prediction. Different structure prediction methods, including homology modeling, fold recognition (FR)/protein threading, ab initio/de novo approaches, and hybrid techniques involving multiple types of approaches, are introduced in a historical context. The progress of the field as a whole, especially in the threading/FR area, as reflected by the CASP/CAFASP contests, is reviewed. At the end of the chapter, we discuss the challenging issues ahead in the field of protein structure prediction.
As a protein evolves, not every part of the amino acid sequence has an equal probability of being deleted or for allowing insertions, because not every amino acid plays an equally important role in maintaining the protein structure. However the most prevalent models in fold recognition methods treat every amino acid deletion and insertion as equally probable events. We have analyzed the alignment patterns for homologous and analogous sequences to determine patterns of insertion and deletions, and used that information to determine the statistics of insertions and deletions for different amino acids of a target sequence. We define these patterns as Insertion/Deletion (Indel) Frequency Arrays (IFA). By applying IFA to the protein threading problem, we have been able to improve the alignment accuracy, especially for proteins with low sequence identity.
The major pathological hallmark of amyloid diseases is the presence of extracellular amyloid deposits. Serum amyloid A (SAA) is an apolipoprotein primarily produced in the liver. Serum protein levels can increase one thousandfold after inflammation. SAA is the precursor to the amyloid A protein found in deposits of systemic amyloid A amyloid (AA or reactive amyloid) in both mouse and human. To study the factors necessary for cerebral amyloid formation, we have created a transgenic mouse that expresses the amyloidogenic mouse Saa1 protein in the brain. Using the synapsin promoter to drive expression of the Saa1 gene, the brains of transgenic mice expressed both RNA and protein. Under noninflammatory conditions, transgenic mice do not develop AA amyloid deposits in the brain; however, induction of a systemic acute-phase response in transgenic mice enhanced amyloid deposition. This deposition was preceded by an increase in cytokine levels in the brain, suggesting that systemic inflammation may be a contributing factor to the development of cerebral amyloid. The nonsteroidal anti-inflammatory agent indomethacin reduced inflammation and protected against the deposition of AA amyloid in the brain. These studies indicate that inflammation plays an important role in the process of amyloid deposition, and inhibition of inflammatory cascades may attenuate amyloidogenic processes, such as Alzheimer's disease.
As a protein evolves, not every part of the amino acid sequence has an equal probability of being deleted or for allowing insertions, because not every amino acid plays an equally important role in maintaining the protein structure. However, the most prevalent models in fold recognition methods treat every amino acid deletion and insertion as equally probable events. We have analyzed the alignment patterns for homologous and analogous sequences to determine patterns of insertion and deletion, and used that information to determine the statistics of insertions and deletions for different amino acids of a target sequence. We define these patterns as insertion/deletion (indel) frequency arrays (IFAs). By applying IFAs to the protein threading problem, we have been able to improve the alignment accuracy, especially for proteins with low sequence identity. We have also demonstrated that the application of this information can lead to an improvement in fold recognition.
Serum amyloid A (SAA) proteins are acute-phase apolipoproteins that are associated with high-density lipoprotein (HDL) particles. SAA proteins are precursors to secondary amyloid fibril proteins and under certain conditions of chronic or recurrent inflammation these proteins are deposited as amyloid fibrils. Of two isotypes found in mouse, SAA1.1 and SAA2.1, only SAA1.1 is deposited into amyloid. The CE/Jmouse is unique, in that the only isoform identified is a hybrid between SAA1.1 and SAA2.1 and the mouse does not show amyloid deposition. In the rat, a deletion in the SAA1/SAA2 gene is associated with the absence of protein in the plasma and subsequently no amyloid deposition is detected. We have generated adenoviral vectors to study the expression of SAA proteins on HDL metabolism and amyloid formation. Injection of SAA viruses into rats resulted in expression of the mouse SAA proteins in the plasma with specific association of the SAA with HDL particles. The induction of SAA proteins was comparable to that seen in mice presented with the inflammatory agent, bacterial lipopolysaccharide (LPS). Adenoviral induced SAA levels were maintained for up to several weeks without a significant decrease in SAA expression. Injection of rats with the mouse SAA 1.1 adenoviral vector, followed by amyloid enhancing factor (AEF) and silver nitrate resulted in the deposition of amyloid fibrils in the spleen. After 2 weeks, amyloid could be detected in other tissues, including the heart, liver, kidneys and lungs. When animals were injected with null or the SAA2.2 virus no amyloid was detected. These studies demonstrate that the inability of the rat to develop AA amyloid is due to the lack of synthesizing an amyloidogenic SAA protein. Furthermore, the expression of the adenoviral SAA protein from the liver and incorporation onto HDL particles further supports the hypothesis that AA amyloid is derived from circulating SAA protein. The ease of use of the adenoviral vectors and the rat provide an excellent model to study the function of SAA proteins.
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