Molecular basis for retinol binding by serum amyloid A during infection.

Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization. In infected mice, SAA proteins circulate in association with the vitamin A derivative retinol, suggesting that SAAs transport retinol during infection. Here we illuminate a structural basis for the retinol-SAA interaction. In the bloodstream of infected mice, most SAA is complexed with high-density lipoprotein (HDL). However, we found that the majority of the circulating retinol was associated with the small fraction of SAA proteins that circulate without binding to HDL, thus identifying free SAA as the predominant retinol-binding form in vivo. We then determined the crystal structure of retinol-bound mouse SAA3 at a resolution of 2.2 Å. Retinol-bound SAA3 formed a novel asymmetric trimeric assembly that was generated by the hydrophobic packing of the conserved amphipathic helices α1 and α3. This hydrophobic packing created a retinol-binding pocket in the center of the trimer, which was confirmed by mutagenesis studies. Together, these findings illuminate the molecular basis for retinol transport by SAA proteins during infection.

Serum Amyloid A (SAA) Proteins.

As normal constituents of blood serum, the Serum Amyloid A (SAA) proteins are small (104 amino acids in humans) and remarkably well-conserved in mammalian evolution. They are synthesized prominently, but not exclusively, in the liver. Fragments of SAA can associate into insoluble fibrils (called "amyloid") characteristic of "secondary" amyloid disease in which they can interrupt normal physiology and lead to organ failure. SAA proteins comprise a family of molecules, two members of which (SAA1 and SAA2) are (along with C-reactive protein, CRP) the most prominent members of the acute phase response (APR) during which their serum levels rise dramatically after trauma, infection and other stimuli. Biologic function (s) of SAA are unresolved but features are consistent with a prominent role in primordial host defense (including the APR ). SAA proteins are lipophilic and contribute to high density lipoproteins (HDL) and cholesterol transport. SAA proteins interact with specific receptors and have been implicated in tissue remodeling through metalloproteinases, local tissue changes in atherosclerosis, cancer metastasis, lung inflammation, maternal-fetal health and intestinal physiology. Molecular details of some of these are emerging.

Aggregation of Mouse Serum Amyloid A Protein Was Promoted by Amyloid-Enhancing Factors with the More Genetically Homologous Serum Amyloid A.

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-ß structure accumulate and are deposited extracellularly in organs, resulting in chronic inflammatory diseases and infections. The incidence of AA amyloidosis is high in humans and several animal species. Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis. Amyloid enhancing factor (AEF) serves as a seed for fibril formation and shortens the onset of AA amyloidosis sharply. In this study, we examined whether AEFs extracted and purified from five animal species (camel, cat, cattle, goat, and mouse) could promote mouse SAA (mSAA) protein aggregation in vitro using quantum-dot (QD) nanoprobes to visualize the aggregation. The results showed that AEFs shortened and promoted mSAA aggregation. In addition, mouse and cat AEFs showed higher mSAA aggregation-promoting activity than the camel, cattle, and goat AEFs. Interestingly, homology analysis of SAA in these five animal species revealed a more similar amino acid sequence homology between mouse and cat than between other animal species. Furthermore, a detailed comparison of amino acid sequences suggested that it was important to mSAA aggregation-promoting activity that the 48th amino acid was a basic residue (Lys) and the 125th amino acid was an acidic residue (Asp or Glu). These data imply that AA amyloidosis exhibits higher transmission activity among animals carrying genetically homologous SAA gene, and may provide a new understanding of the pathogenesis of amyloidosis.

COOH-terminal SAA1 peptides fail to induce chemokines but synergize with CXCL8 and CCL3 to recruit leukocytes via FPR2.

A natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4 Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.

Structural Basis for Vital Function and Malfunction of Serum Amyloid A: an Acute-Phase Protein that Wears Hydrophobicity on Its Sleeve.

PURPOSE OF REVIEW: This review addresses normal and pathologic functions of serum amyloid A (SAA), an enigmatic biomarker of inflammation and protein precursor of AA amyloidosis, a life-threatening complication of chronic inflammation. SAA is a small, highly evolutionarily conserved acute-phase protein whose plasma levels increase up to one thousand-fold in inflammation, infection, or after trauma. The advantage of this dramatic but transient increase is unclear, and the complex role of SAA in immune response is intensely investigated. This review summarizes recent advances in our understanding of the structure-function relationship of this intrinsically disordered protein, outlines its newly emerging beneficial roles in lipid transport and inflammation control, and discusses factors that critically influence its misfolding in AA amyloidosis. RECENT FINDINGS: High-resolution structures of lipid-free SAA in crystals and fibrils have been determined by x-ray crystallography and electron cryo-microscopy. Low-resolution structural studies of SAA-lipid complexes, together with biochemical, cell-based, animal model, genetic, and clinical studies, have provided surprising new insights into a wide range of SAA functions. An emerging vital role of SAA is lipid encapsulation to remove cell membrane debris from sites of injury. The structural basis for this role has been proposed. The lysosomal origin of AA amyloidosis has solidified, and its molecular and cellular mechanisms have emerged. Recent studies have revealed molecular underpinnings for understanding complex functions of this Cambrian protein in lipid transport, immune response, and amyloid formation. These findings help guide the search for much-needed targeted therapies to block the protein deposition in AA amyloidosis.

Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH and contribute to the pathogenesis of reactive amyloidosis.

Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5-4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with ∼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to ß-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5-4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.

Suppression of Lipopolysaccharide-Induced Inflammatory Response by Fragments from Serum Amyloid A.

Serum amyloid A (SAA) is known as an acute-phase protein and a biomarker for inflammatory diseases. Published studies have shown that SAA possesses proinflammatory cytokine-like activity and is chemotactic for phagocytes, but the structural basis for these activities remains unidentified. In this article, we report that truncated SAA1 proteins lacking N- and C-terminal sequences exhibit reduced proinflammatory activity and strongly suppress LPS-induced expression of IL-1ß, IL-6, and TNF-α in macrophages. A truncated SAA1 containing aa 11-58 was examined further and found to facilitate p38 MAPK phosphorylation while reducing LPS-stimulated phosphorylation of ERK and JNK. In LPS-challenged mice, aa 11-58 reduced the severity of acute lung injury, with significantly less neutrophil infiltration in the lungs and attenuated pulmonary expression of IL-1ß, IL-6, and TNF-α. Coadministration of aa 11-58 markedly improved mouse survival in response to a lethal dose of LPS. A potent induction of IL-10 was observed in a TLR2-dependent, but TLR4-independent, manner in macrophages stimulated with aa 11-58. However, the aa 11-58 fragment of SAA1 was unable to induce chemotaxis or calcium flux through formyl peptide receptor 2. These results indicate that the N- and C-terminal sequences contain structural determinants for the proinflammatory and chemotactic activities of SAA1, and their removal switches SAA1 to an anti-inflammatory role. Given that proteolytic processing of SAA is associated with the pathological changes in several diseases, including secondary amyloidosis, our findings may shed light on the structure-function relationship of SAA1 with respect to its role in inflammation.

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits.

Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions.

Acceleration of amyloid fibril formation by carboxyl-terminal truncation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis. Although the full-length SAA is 104 amino acids long, the C-terminal-truncated SAA lacking mainly residues 77-104 is predominantly deposited in AA amyloidosis. Nevertheless, the amyloid fibril formation of such truncated forms of human SAA has never been investigated. In the present study, we examined the effect of C-terminal truncation on amyloid fibril formation of human SAA induced by heparan sulfate (HS). Circular dichroism (CD) measurements demonstrated that the C-terminal truncation induces a reduced α-helical structure of the SAA molecule. HS-induced increases in thioflavin T fluorescence for SAA (1-76) peptide and less significant increases for full-length SAA were observed. CD spectral changes of SAA (1-76) peptide but not full-length SAA were observed when incubated with HS, although the spectrum was not typical for a ß-structure. Fourier transform infrared experiments clearly revealed that SAA (1-76) peptide forms a ß-sheet structure. Transmission electron microscopy revealed that short fibrillar aggregates of SAA (1-76) peptides, which became longer with increasing peptide concentrations, were observed under conditions in which full-length SAA scarcely formed fibrillar aggregates. These results suggested that the C-terminal truncation of human SAA accelerates amyloid fibril formation.

Dynamic changes of the composition of plasma HDL particles in patients with cardiac disease: Spotlight on sphingosine-1-phosphate/serum amyloid A ratio.

Several epidemiological studies reported an inverse relationship between plasma high-density lipoprotein (HDL) cholesterol levels and atherosclerotic cardiovascular disease (ASCVD). However, therapeutic interventions targeted at raising HDL-cholesterol failed to improve cardiovascular outcomes, suggesting that HDL components distinct from cholesterol may account for the anti-atherothrombotic effects attributed to this lipoprotein. Sphingosine-1-phosphate (S1P) and the acute phase protein serum amyloid A (SAA) have been identified as integral constituents of HDL particles. Evidence suggests that S1P and SAA levels within HDL particles may be affected by inflammation and oxidative stress, which are coexisting processes underlying ASCVD. Because SAA, an inflammation-related marker, and S1P, an anti-atherothrombotic marker, have relatively clear opposite characteristics among the HDL-associated proteins, the approach of assessing the two markers simultaneously may provide new insights in clinical practice (S1P/SAA Index). This review focuses on evidence in support of the concept that the S1P/SAA Index may affect the HDL atheroprotective properties and may, therefore represent a potential target for therapeutic interventions.

Serum amyloid A self-assembles with phospholipids to form stable protein-rich nanoparticles with a distinct structure: A hypothetical function of SAA as a "molecular mop" in immune response.

Serum amyloid A (SAA) is an acute-phase protein whose action in innate immunity and lipid homeostasis is unclear. Most circulating SAA binds plasma high-density lipoproteins (HDL) and reroutes lipid transport. In vivo SAA binds existing lipoproteins or generates them de novo upon lipid uptake from cells. We explored the products of SAA-lipid interactions and lipoprotein remodeling in vitro. SAA complexes with palmitoyl-oleoyl phosphocholine (POPC) were analyzed for structure and stability using circular dichroism and fluorescence spectroscopy, electron microscopy, gel electrophoresis and gel filtration. The results revealed the formation of 8-11nm lipoproteins that were∼50% α-helical and stable at near-physiological conditions but were irreversibly remodeled at Tm∼52°C. Similar HDL-size nanoparticles formed spontaneously at ambient conditions or upon thermal remodeling of parent lipoproteins containing various amounts of proteins and lipids, including POPC and cholesterol. Therefore, such HDL-size particles formed stable kinetically accessible structures in a wide range of conditions. Based on their size and stoichiometry, each particle contained about 12 SAA and 72 POPC molecules, with a protein:lipid weight ratio circa 2.5:1, suggesting a structure distinct from HDL. High stability of these nanoparticles and their HDL-like size suggest that similar lipoproteins may form in vivo during inflammation or injury when SAA concentration is high and membranes from dead cells require rapid removal. We speculate that solubilization of membranes by SAA to generate lipoproteins in a spontaneous energy-independent process constitutes the primordial function of this ancient protein, providing the first line of defense in clearing cell debris from the injured sites.

Serum amyloid A proteins take retinol for a ride.

Vitamin A plays pleiotropic roles in the immune system. A recent eLife paper by Hooper and colleagues shows that hepatic and intestinal serum amyloid A proteins, which are induced in response to infection, can transport vitamin A metabolites to tissues and thus impact immune responses both locally and systemically.

Recombinant expression of Epinephelus lanceolatus serum amyloid A (ElSAA) and analysis of its macrophage modulatory activities.

Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.

Cloning, expression analysis, and antibacterial propertiesof three serum amyloid A in common carp (Cyprinus carpio).

Three serum amyloid A (SAA) genes were identified from the common carp (Cyprinus carpio) by PCR and RT-PCR. Considering both direction and sequence similarity with mammal's orthologs, they were named CcSAA3a, CcSAA3b and CcSAA1. CcSAA3b and CcSAA1 are adjacent on contig LHQP01017858, suggesting that the prototype of or the simplest SAA multigene family have occurred in common carp. A phylogenetic analysis of the SAAs indicated that the fish SAAs were closer to those of invertebrates and Ornithorhynchus anatinus, a primitive mammal, than to mammalian SAAs. Quantitative real-time RT-PCR results displayed different expression profiles of three CcSAAs. The CcSAA3a was detected in all tested tissues, and was most abundant in the muscle; CcSAA3b was predominately expressed in the intestine and liver, and CcSAA1 in the skin. The expression level of CcSAA3a was higher than that of CcSAA3b and CcSAA1 in most tissues. Stimulation with Aeromonas hydrophila dramatically induced the expression of the three CcSAAs in all examined tissues, especially in the liver. Like Epinephelus coioides SAA, all of three rCcSAA fusion proteins could bind to both Gram-negative bacteria (A. hydrophila and E. coli) and Gram-positive bacterium (S. aureus), playing a role in the identification of bacteria. However, only rCcSAA3a showed significantly anti-A. hydrophila and anti-E. coli in vitro antibacterial activity assays. These results suggested that the three CcSAAs were in functional differentiation and play significant roles in the innate immunity of common carp.

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis.

Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation.

Paradoxical effects of SAA on lipoprotein oxidation suggest a new antioxidant function for SAA.

Oxidative stress and inflammation, which involve a dramatic increase in serum amyloid A (SAA) levels, are critical in the development of atherosclerosis. Most SAA circulates on plasma HDL particles, altering their cardioprotective properties. SAA-enriched HDL has diminished anti-oxidant effects on LDL, which may contribute to atherogenesis. We determined combined effects of SAA enrichment and oxidation on biochemical changes in HDL. Normal human HDLs were incubated with SAA, oxidized by various factors (Cu2+, myeloperoxidase, H2O2, OCl-), and analyzed for lipid and protein modifications and biophysical remodeling. Three novel findings are reported: addition of SAA reduces oxidation of HDL and LDL lipids; oxidation of SAA-containing HDL in the presence of OCl- generates a covalent heterodimer of SAA and apoA-I that resists the release from HDL; and mild oxidation promotes spontaneous release of proteins (SAA and apoA-I) from SAA-enriched HDL. We show that the anti-oxidant effects of SAA extend to various oxidants and are mediated mainly by the unbound protein. We propose that free SAA sequesters lipid hydroperoxides and delays lipoprotein oxidation, though much less efficiently than other anti-oxidant proteins, such as apoA-I, that SAA displaces from HDL. These findings prompt us to reconsider the role of SAA in lipid oxidation in vivo.

Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice.

The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism.

Contrasting increased levels of serum amyloid A in patients with three different bone sarcomas: An indicator of tumor malignancy?

Sarcoma is a malignant tumor that originates from the bone or soft tissue. In this study, abundances of serum amyloid A (SAA) in patients with pleomorphic sarcoma (PS), chondrosarcoma (CS), and osteosarcoma (OS) were analyzed and compared with those from their respective age-matched healthy control subjects. Results obtained from our analysis by 2DE showed that the levels of SAA were markedly elevated in patients with PS and OS, which are highly metastatic, while in patients with CS, which is a less aggressive sarcoma, the increase appeared less pronounced. A similar trend of altered abundances was also observed when the levels of SAA in the subjects were estimated using Western blot, ELISA, and multiple-reaction monitoring analyses. Absolute quantification using multiple-reaction monitoring further demonstrated that the increased abundance of SAA in patients with PS, OS, and CS was mainly attributed to isoform SAA1. In view of the different degrees of tumor malignancy in PS, OS, and CS, our data suggest their apparent correlation with the levels of SAA in the patients.

Thermal transitions in serum amyloid A in solution and on the lipid: implications for structure and stability of acute-phase HDL.

Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma HDL. SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace a substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the nonexchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies in acute-phase HDL. Second, we report an α-helix folding/unfolding transition in SAA in the presence of lipid at near-physiological temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.

Structural studies of the C-terminal 19-peptide of serum amyloid A and its Pro → Ala variants interacting with human cystatin C.

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose concentration in serum increases markedly following a number of chronic inflammatory and neoplastic diseases. Prolonged high SAA level may give rise to reactive systemic amyloid A (AA) amyloidosis, where the N-terminal segment of SAA is deposited as amyloid fibrils. Besides, recently, well-documented association of SAA with high-density lipoprotein or glycosaminoglycans, in particular heparin/heparin sulfate (HS), and specific interaction between SAA and human cystatin C (hCC), the ubiquitous inhibitor of cysteine proteases, was proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, a hCC binding site in the SAA sequence was determined as SAA(86-104). The role of this SAA C-terminal fragment as a ligand-binding locus is still not clear. It was postulated important in native SAA folding and in pathogenesis of AA amyloidosis. In the search of conformational details of this SAA fragment, we did its structure and affinity studies, including its selected double/triple Pro → Ala variants. Our results clearly show that the SAA(86-104) 19-peptide has rather unordered structure with bends in its C-terminal part, which is consistent with the previous results relating to the whole protein. The results of affinity chromatography, fluorescent ELISA-like test, CD and NMR studies point to an importance of proline residues on structure of SAA(86-104). Conformational details of SAA fragment, responsible for hCC binding, may help to understand the objective of hCC-SAA complex formation and its importance for pathogenesis of reactive amyloid A amyloidosis.