Recent data concerning the effect of soybean 7S globulin subunits on the upregulation of LDL receptors in Hep G2 cells identified the alpha' subunit as the candidate responsible for this biological effect. In vivo evaluation of this subunit on cholesterol homeostasis was hampered by the lack of suitable amounts of alpha' chain. A novel separation procedure allowed us to investigate the effects of alpha' subunit administration on plasma cholesterol and triglyceride levels, as well as on the activity of liver beta-VLDL receptors of rats fed a hypercholesterolemic (HC) diet. Rats were divided into 9 groups fed the following diets for 28 d: standard diet; HC diet; HC diets + 5, 10, and 20 mg/(kg body weight. d) of alpha' subunit; HC diets + 50, 100, and 200 mg/(kg body weight. d) of soybean 7S globulin; HC diet + 200 mg/(kg body weight. d) clofibrate. The highest dose of the alpha' subunit decreased plasma cholesterol and triglycerides 36 and 34%, respectively, in rats fed the HC diet; 10-fold amounts clofibrate reduced plasma cholesterol and triglycerides 38 and 41%. The activity of liver beta-VLDL receptors of rats fed the HC diet with the highest dose of the alpha' subunit had a 96% increase in binding compared with the HC diet group, thus restoring the receptor activity to that of rats fed the standard diet. These results represent the first in vivo evidence of both the plasma lipid-lowering properties and the upregulation of liver beta-VLDL receptors induced by the soybean alpha' subunit.

Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process. A prototypic example is the D76N mutant of beta-2 microglobulin (β2m) that displays an aggressive aggregation propensity. Here we investigate the dynamics of β2m by X-ray crystallography, solid-state NMR, and molecular dynamics simulations to unveil the effects of the D76N mutation. Taken together, our data highlight the presence of minor disordered substates in crystalline β2m. The destabilization of the outer strands of D76N β2m accounts for the increased aggregation propensity. Furthermore, the computational modeling reveals a network of interactions with residue D76 as a keystone: this model allows predicting the stability of several point mutants. Overall, our study shows how the study of intrinsic dynamics in crystallo can provide crucial answers on protein stability and aggregation propensity. The comprehensive approach here presented may well be suited for the study of other folded amyloidogenic proteins.

Light chain amyloidosis (AL), the most common systemic amyloidosis, is caused by the overproduction and the aggregation of monoclonal immunoglobulin light chains (LC) in target organs. Due to genetic rearrangement and somatic hypermutation, virtually, each AL patient presents a different amyloidogenic LC. Because of such complexity, the fine molecular determinants of LC aggregation propensity and proteotoxicity are, to date, unclear; significantly, their decoding requires investigating large sets of cases. Aiming to achieve generalizable observations, we systematically characterised a pool of thirteen sequence-diverse full length LCs. Eight amyloidogenic LCs were selected as responsible for severe cardiac symptoms in patients; five non-amyloidogenic LCs were isolated from patients affected by multiple myeloma. Our comprehensive approach (consisting of spectroscopic techniques, limited proteolysis, and X-ray crystallography) shows that low fold stability and high protein dynamics correlate with amyloidogenic LCs, while hydrophobicity, structural rearrangements and nature of the LC dimeric association interface (as observed in seven crystal structures here presented) do not appear to play a significant role in defining amyloid propensity. Based on the structural and biophysical data, our results highlight shared properties driving LC amyloid propensity, and these data will be instrumental for the design of synthetic inhibitors of LC aggregation.

α-Synuclein is a presynaptic protein associated to Parkinson’s disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson’s disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

Structural changes of starch and proteins in rice pasta were investigated as a function of raw-materials and pasta-making conditions, and their impact on cooking behaviour and glycaemic index was assessed. Rice pasta was prepared from untreated or parboiled rice flour by conventional extrusion or by extrusion-cooking. Starch structure was studied by assessing starch accessibility to specific enzymes (α-amylase and pullulanase), and by evaluating the molecular properties of fragments from enzymatic action. Protein solubility in presence/absence of chaotropes and accessibility of protein cysteine thiols allowed to evaluate the intensity and nature of inter-protein interactions. Parboiling stiffens the protein network in rice flour and makes starch more accessible to hydrolysis. Pasta-making induced further changes in the starch structure, that were most evident in pasta made from untreated rice and were mainly related to the amylopectin fraction. Thus, the interplay among structural modifications on starch and/or proteins affects the features of products.

b2 microglobulin (b2m) is the light chain of class-I major histocompatibility complex (MHC-I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of b2m aggregation, several b2m mutants have been previously studied. Among these, three single-residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect b2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild-type (w.t.) b2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray-ionization mass spectrometry (ESI-MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE-loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, b2m DE-loop mutations display long-range effects, affecting stability and structural properties of the native protein and its low-pH intermediate. The evidence presented here hints to a crucial role played by the DE-loop in determining the overall properties of native and partially folded b2m.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. a b s t r a c tThe antimicrobial proteins lysozyme and lactoferrin were incorporated into paper containing carboxymethyl cellulose, that allowed non-covalent binding of the positively charged proteins to the paper matrix. More than 60 percent of the proteins added alone or in combination during the papermaking process were released in buffered saline. The released proteins retained their structural and functional features, indicating that the papermaking process did not affect their structure. The antimicrobial activity on common food contaminants was also retained in the released protein, and a synergism between the two proteins was evident in tests carried out with paper containing both proteins against Listeria.Tests on thin meat slices laid on paper sheets containing either or both antimicrobial proteins indicated that lysozyme was most effective in preventing growth of this particular microbiota.

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His 69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (ϳ3 versus ϳ5 M, respectively), and the mutant protein had a melting temperature ϳ10 -15°C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 M urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.Bacterial cholesterol oxidase (CO, EC 1.1.3.6) is a monomeric bifunctional FAD-containing flavoenzyme that catalyzes the oxidation of 3␤-hydroxysteroids and the isomerization of the intermediate, ⌬ 5-6 -ene-3␤-ketosteroid (cholest-5-en-3-one) to produce ⌬ 3-4 -ene-3␤-ketosteroid (cholest-4-en-3-one). Bacteria that produce CO can be classified into two classes: non-pathogenic (e.g. Streptomyces and fast-growing Mycobacteria; these bacteria utilize cholesterol as their carbon source) and pathogenic bacteria (e.g. Rhodococcus equi and slow-growing Mycobacteria). Pathogenic bacteria require CO for infection of the host macrophage probably because of its ability to alter the physical structure of the membrane by converting cholesterol into cholest-4-en-3-one.The crystal structure of COs belonging to two distinct structural classes (1) have been solved by the Vrielink laboratory (2-4). Members classified as type I, such as those from Streptomyces sp. SA-COO and R. equi (5) have their cofactor tightly but non-covalently bound to the enzyme. A second CO from Brevibacterium sterolicum classified as type II, is completely different. In this enzyme the cofactor is covalently linked to the protein via a bond between the 8-methyl group of the isoalloxazine ring and the ND1 atom of His 69 .Although COs of class I an...