Serum Albumin Prevents Protein Aggregation and Amyloid Formation and Retains Chaperone-like Activity in the Presence of Physiological Ligands Thomas E. Finn, Andrea C. Nunez, Margaret Sunde1, and Simon B. Easterbrook-Smith

School of Molecular Bioscience and Discipline of Pharmacology, The University of Sydney, Sydney,Australia. JBC Papers.

PRESENTED BY: MOHD SHAMOON ASMAT

INTRODUCTION

Serum albumin - Most abundant protein in the serum of mammals.

Functions: Transport , solubility of ligands(Cu2+,Ca2+ , FFAs)

Marini et al observed that BSA is able to protect no. of enzymes from thermal agg and inactivation

Potential role of chaperone proteins in the pathogenesis and treatment of neurodeg. diseases has become a topic of significant interest

Groups studying amyloid peptide (Aβ) amyloid formation have shown that serum albumin tightly binds Aβ inhibits Aβ fibril formation .

OBJECTIVE

In this paper, the chaperone-like activity of serum albumin is characterized at physiological conc of both serum albumin and imp ligands.

At physiological conc, molecular crowding effects become significant,may accelerate or inhibit protein agg.

Using established Assays, the antiagg and anti-amyloid properties of serum albumin are demonstrated, and evidence is provided that these properties arise from specific interactions b/w serum albumin and its client proteins.

MATERIALS AND METHODS

MATERIALS

Metal-free BSA

NaCl/HEPES Buffer(250mM NaCl , 30mM HEPES pH7.4)

EDTA

ADH

Insulin

Wild type TTR & L55P Mutant TTR

DiThioThreitol(DTT)

Free Fatty Acids(Palmitic,Stearic,Oleic and Linoleic 683uM)

METHODS

Uv-spectroscopy

ELISA

Fluoroscence Spectroscopy

Centrifuge filter device

Gel Filtration Chromatography

RESULTS & DISCUSSION

BSA displays chaperone-like activity at physiological concentration towards ADH and Insulin.

This activity was assessed by observing the influence of BSA on the agg of two proteins: alcohol dehydrogenase and insulin.

The chaperone-like effect was titratable

BSA was a more potent inhibitor of insulin aggregation than it was for ADH At low concentrations, BSA had an unusual effect on ADH agg and may be possible

that low level crowding is affecting the folding equilibrium

With ADH with Insulin

> Wild type TTR and lysozyme were employed as controls Physiological conc of TTR did not inhibit ADH agg nor did they alter insulin agg Conversely, lysozyme actually enhanced aggregation of both proteins

With Transthyretin With Lysozyme

BSA in the presence of Physiologicalligands

Three important BSA ligands: Cu2+,Ca2+ , FFAs were investigated for any effect on the chaperone-like activity of BSA.

Target proteins were agg with [BSA] kept constant

Ca2+ was varied 0.5-4 times the physiological level relative to [BSA] and absolute physiological levels were also included.In a similar manner, the effect of Cu2+ was varied.

Neither Cu2+ nor Ca2 caused any measurable interference with the chaperone-like activity of BSA toward either ADH or insulin

Because hydrophobic interactions are known to be imp the effect ofchanging FFA concentrations on BSA chaperone-like activity was investigated.

Palmitic, stearic, oleic, and linoleic acids together make up 90% of circulating FFAsand 80% of the FFAs bound to serum albumin.

Titrating FFAs into the ADH agg rxn resulted in increase of agg and reduction in the protective effect of BSA

BSA inhibits aggregation and amyloid formation by WT-TransThyretin and L55P-Ttr

To assess whether BSA exhibited anti-amyloid activity, its effect on Ttr agg and amyloid formation was assessed. WT-TTR and L55P-TTR at physiological levels were induced to form amyloid by incubation at 37 °C in acetate buffer at pH 4.2 and 5.2, resp, in the absence or presence of BSA.

Total aggregation was measured and Formation of amyloid was determined by induction of ThT fluorescence

WT-Ttr Amyloid formation was carried out in the absence and presence of BSA.The aliquots were removed at the specified times and were assessed for A350 ( net agg) and ThT fluorescence (amyloid formation)

The effect of BSA on L55P-TTR was similar although this effect was achieved at lower concentrations of BSA.Analysis of the T50 emphasized the diff b/w the WT and L55P-TTRBSA not only reduces agg and amyloid formation by WT and L55P-TTR in a dose-dependent manner but also titratably slows the formation of agg and amyloid by the L55P mutant

BSA displayed preferential binding to stressed ADH and insulin over the native form

ELISAs were performed to assess whether BSA shows similar preferential binding to denatured proteins.

BSA forms stable high molecular weight species with stressed target proteins

Size exclusion chromatography was used to determine whetherBSA also preferentially binds stressed proteins to form HMW complexesand also whether FFAs inhibit the formation of such complexes.

Target proteins were stressed in the presence or absence of BSAwith or without FFAs, and soluble fractions were analysedby size exclusion chromatography .

Solutions of individual proteins or mixtures of BSA, FFAs and target protein (ADH or insulin ) were left in their native state or stressed.

The above results are in line with both the greater ability of BSA to protect insulin from agg and differing affinities of BSA for stressed/unstressed AHD and insulin

Aggregation of human serum under physiological shear and Heat Stress

To determine whether serum albumin maintains chaperone-like properties in vivo, human plasma was induced to aggr using physiologically relevant stresses.

Plasma depleted of albumin was compared with plasma controls that were either diluted to a comparable level of non albumin protein or diluted to the same A280

To ensure that the changed aggregation behavior was a result of the removal of albumin, BSA was reintroduced to albumin-depleted plasma and compared

Next,. To elucidate whether FFA elevations present in normal physiology may alter the chaperone-like activity of serum albumin, additional FFA at a conc 3 times that of physiological levels was added to undiluted plasma, and the agg behavior was compared with native plasma

A depletion of albumin from plasma leads to increased aggregation under physiologically relevant stresses. , serum albumin was depleted from plasma.Depletion of serum albumin results in a significant increase in aggregation of plasma.

CONCLUSION Serum albumin when present at physiological levels, reduces agg or amyloid

formation of its client proteins.

Chaperone-like activity is maintained in the presence of Ca2, Cu2, and FFAs.

Presence of Ca2 did not observably alter the chaperone-like properties of serum albumin Effect of a wide range of Cu2 conc was assessed, yet no significant effect on serum albumin activity was observed. FFAs alter the chaperone-like activity of serum albumin toward both client proteins.

Insulin was protected far more effectively than ADH.

Results show that plasma exposed to these physiological stresses agg to a greater extent when depleted of serum albumin than when serum albumin is present.

The chaperone-like activity of serum albumin suggests it protects against protein misfolding and agg.

This may have implications for protein misfolding disorders of the extracellular compartment

JAZAKALLLAH