Removal Of Protein Aggregates Research Articles

Cullins Keep Autophagy under Control

Autophagy removes protein aggregates, damaged organelles, and intracellular pathogens from cells and helps to recycle lipids and protein building blocks. However, regulatory mechanisms of the initial, rapid response are largely unknown. In this issue of Developmental Cell, Antonioli et al. (2014) provide mechanistic insights into autophagy induction and termination by Cullin E3 ligases.

Mechanisms and biological functions of autophagy in diseased and ageing kidneys.

Autophagy degrades pathogens, altered organelles and protein aggregates, and is characterized by the sequestration of cytoplasmic cargos within double-membrane-limited vesicles called autophagosomes. The process is regulated by inputs from the cellular microenvironment, and is activated in response to nutrient scarcity and immune triggers, which signal through a complex molecular network. Activation of autophagy leads to the formation of an isolation membrane, recognition of cytoplasmic cargos, expansion of the autophagosomal membrane, fusion with lysosomes and degradation of the autophagosome and its contents. Autophagy maintains cellular homeostasis during stressful conditions, dampens inflammation and shapes adaptive immunity. A growing body of evidence has implicated autophagy in kidney health, ageing and disease; it modulates tissue responses during acute kidney injuries, regulates podocyte homeostasis and protects against age-related renal disorders. The renoprotective functions of autophagy in epithelial renal cells and podocytes are mostly mediated by the clearance of altered mitochondria, which can activate inflammasomes and apoptosis, and the removal of protein aggregates, which might trigger inflammation and cell death. In translational terms, autophagy is undoubtedly an attractive target for developing new renoprotective treatments and identifying markers of kidney injury.

Autophagy and heart disease: implications for cardiac ischemia-reperfusion damage.

Survival of myocytes and mesenchymal cells in the heart is tightly regulated by a number of adaptive processes that are invoked with the changes that occur within the parenchyma and stroma. Autophagy is implicated in cellular housekeeping duties and maintenance of the integrity of the intracellular milieu by removal of protein aggregates and damaged organelles, whereas under pathophysiological conditions, the chronic up-regulation of autophagy may lead to significant disturbance of homeostatic conditions. Nonetheless, the role of autophagy in heart disease in the context of cardiac ischemia-reperfusion injury is currently unclear. This review will focus upon the role of autophagy as it pertains to ischemia reperfusion damage in the heart.

Autophagy: Emerging Therapeutic Target for Diabetic Nephropathy

Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis and to keep cells healthy. The accumulation of damaged proteins and organelles induced by hyperglycemia and other metabolic alterations is strongly associated with the development of diabetic nephropathy. Autophagy is up-regulated under conditions of calorie restriction and environmental stress, such as oxidative stress and hypoxia in proximal tubular cells, and occurs even under normal conditions in podocytes. These findings have led to our hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy, a hypothesis increasingly supported by experimental evidence. To date, however, the exact role of autophagy in diabetic nephropathy has not been fully revealed. This article therefore reviews recent findings and provides perspectives on the involvement of autophagy in diabetic nephropathy.

Excitation BolsTORs Motor Neurons in ALS Mice

It is unclear why motor neurons selectively degenerate in amyotrophic lateral sclerosis (ALS). Saxena etal. (2013) demonstrate that excitation of motor neurons can prevent their demise in a mouse model of inherited ALS by a mechanism involving the mTOR pathway.

Aldosterone and angiotensin II induce protein aggregation in renal proximal tubules

Renal tubules are highly active transporting epithelia and are at risk of protein aggregation due to high protein turnover and/or oxidative stress. We hypothesized that the risk of aggregation was increased upon hormone stimulation and assessed the state of the intracellular protein degradation systems in the kidney from control rats and rats receiving aldosterone or angiotensin II treatment for 7 days. Control rats formed both aggresomes and autophagosomes specifically in the proximal tubules, indicating a need for these structures even under baseline conditions. Fluorescence sorted aggresomes contained various rat keratins known to be expressed in renal tubules as assessed by protein mass spectrometry. Aldosterone administration increased the abundance of the proximal tubular aggresomal protein keratin 5, the ribosomal protein RPL27, ataxin-3, and the chaperone heat shock protein 70-4 with no apparent change in the aggresome–autophagosome markers. Angiotensin II induced aggregation of RPL27 specifically in proximal tubules, again without apparent change in antiaggregating proteins or the aggresome–autophagosome markers. Albumin endocytosis was unaffected by the hormone administration. Taken together, we find that the renal proximal tubules display aggresome formation and autophagy. Despite an increase in aggregation-prone protein load in these tubules during hormone treatment, renal proximal tubules seem to have sufficient capacity for removing protein aggregates from the cells.

Mitochondrial Autophagy Involving Renal Injury and Aging Is Modulated by Caloric Intake in Aged Rat Kidneys

BackgroundA high-calorie (HC) diet induces renal injury and promotes aging, and calorie restriction (CR) may ameliorate these responses. However, the effects of long-term HC and CR on renal damage and aging have been not fully determined. Autophagy plays a crucial role in removing protein aggregates and damaged organelles to maintain intracellular homeostasis and function. The role of autophagy in HC-induced renal damage is unknown.MethodsWe evaluated the expression of LC3/Atg8 as a marker of the autophagosome; p62/SQSTM1; polyubiquitin aggregates as markers of autophagy flux; Ambra1, PINK1, Parkin and Bnip3 as markers of mitophagy; 8-hydroxydeoxyguanosine (8-OHdG) as a marker of DNA oxidative damage; and p16 as a marker of organ aging by western blot and immunohistochemical staining in the kidneys of 24-month-old Fischer 344 rats. We also observed mitochondrial structure and autolysosomes by transmission electron microscopy.ResultsExpression of the autophagosome formation marker LC3/Atg8 and markers of mitochondrial autophagy (mitophagy) were markedly decreased in the kidneys of the HC group, and markedly increased in CR kidneys. p62/SQSTM1 and polyubiquitin aggregates increased in HC kidneys, and decreased in CR kidneys. Transmission electron microscopy demonstrated that HC kidneys showed severe abnormal mitochondrial morphology with fewer autolysosomes, while CR kidneys exhibited normal mitochondrial morphology with numerous autolysosomes. The level of 8-hydroxydeoxyguanosine was increased in HC kidneys and decreased in CR kidneys. Markers of aging, such as p16 and senescence-associated-galactosidase, were increased significantly in the HC group and decreased significantly in the CR group.ConclusionThe study firstly suggests that HC diet inhibits renal autophagy and aggravates renal oxidative damage and aging, while CR enhances renal autophagy and ameliorates oxidative damage and aging in the kidneys.

O4‐11‐03: Interaction between the ubiquitin proteasome system, autophagy and progressive tauopathy in vivo

Neurodegenerative diseases including Alzheimer's Disease (AD) and the tauopathy known as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) are characterized with intracellular inclusions of misfolded proteins. Downregulation and dysfunction of either the ubiquitin-proteasome system (UPS) or the autophagic-lysosomal system (A-LS) that clear misfolded proteins and maintain cellular function have been vastly reported in the development of these diseases. Macroautophagy (autophagy), a major degradative pathway of the lysosomal system, plays a significant role in the removal of protein aggregates that are too large, or that cannot be unfolded by chaperone proteins and that are consequently unable to be degraded by the UPS. We hypothesize that in the early stages of tauopathy the UPS is down regulated and is unable to efficiently clear misfolded/ubiquitinated tau species, thus the system becomes overwhelmed as larger aggregates of tau accumulate. We envisage that the A-LS is then upregulated in an effort to compensate for the lost UPS activity and to clear the aggregates, but ultimately both systems fail resulting in accelerated pathology. To investigate the interplay between UPS and A-LS functioning with tau accumulation we use in vivo, ex vivo (acute organotypic slices) and brain homogenate studies of the young and old rTg4510 line that expresses the P301L mutant tau transgene at high levels and develop robust pathology. We demonstrate that accumulation of hyper-phosphorylated tau precedes elevation of ubiquitinated proteins and induction of autophagy markers. The decline in proteasome activity correlates with accumulation of tau aggregates. Additionally, we show that administration of the phosphodiesterase 4 inhibitor, rolipram, elevates proteasome activity, reduces tau levels and improves cognitive performance in rTg4510 mice with mild pathology. In advanced pathology, activation of proteasome failed to reduce tau levels and improves cognitive performance. Our data support the view that tau aggregates perturb the function of UPS in a time-dependent manner and induce autophagy, but insufficiently to clear the aggregates. Moreover, pharmacologic elevation of proteasome activity in vivo and ex vivo of rTg4510 mice reduces tau levels and improves cognitive performance, suggesting a novel therapeutic approach to treat tauopathy disorders including AD and FTD.

Microtubule-associated Protein 1 Light Chain 3 (LC3) Interacts with Bnip3 Protein to Selectively Remove Endoplasmic Reticulum and Mitochondria via Autophagy

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.

Lithium in Multisystem Atrophy: Lack of Efficacy and Safety Issues (P06.073)

Objective: Aim of our study was to determine if lithium was safe and well tolerated in MSA patients. Secondary objectives were the effect of lithium on disease progression, quality of life, and depression. Background Multiple system atrophy (MSA) is a rare, sporadic, progressive neurodegenerative disease. Oligodendrocyte cytoplasmic inclusions of fibrillized alpha-synuclein represent the hallmark of the disease. It was demonstrated that lithium is able to stimulate autophagy, and to efficiently remove protein aggregates, including alpha-synuclein. Design/Methods: We designed a double-blind, placebo-controlled, randomized pilot clinical trial to test the safety and tolerability of 48 weeks of treatment with lithium in MSA. Lithium was administered starting from 150 mg twice-daily. The dose was increased of 300 mg at each visit until lithium serum levels reached 0.9-1.2 mMol/L. The maximum allowed dose was 1500 mg/day. Results: Ten patients were enrolled after informed consent was obtained. Nine patients were randomized, four received lithium and five placebo. In the lithium group one patient died, and the other three abandoned the trial for side effects. Two patients reached the target serum levels of lithium, the other two patients stopped at 0.8, and 0.5 mMol/L when AEs forced them to abandon the trial. One patient of the placebo group died. No other patient retired in the placebo group. An interim analysis was performed one year after the first patient was randomized. The results showed a proportion of trial abandon/death of 100% in the lithium and 20% in the placebo group (p Conclusions: Lithium was overall not well tolerated and caused all of the treated patients to abandon the trial. No conclusions can be drawn on the effects of lithium on secondary endpoints of the study. We do not encourage future studies with lithium in MSA patients. Supported by: “Associazione Italiana per la lotta alle Sindromi Atassiche (AISA) sez. Campania”. Disclosure: Dr. Sacca has nothing to disclose. Dr. Marsili has nothing to disclose. Dr. Brunetti has nothing to disclose. Dr. Carbone has nothing to disclose. Dr. De Michele has nothing to disclose. Dr. Pane has nothing to disclose. Dr. Russo has nothing to disclose. Dr. Salvatore has nothing to disclose. Dr. Tucci has nothing to disclose. Dr. Quarantelli has nothing to disclose. Dr. Filla has nothing to disclose.

Adenovirus Degradation of Cellular Proteins

Eukaryotic cells orchestrate constant synthesis and degradation of intracellular components, including soluble proteins and organelles. The two major intracellular degradation pathways are the ubiquitin/proteasome system and autophagy. Whereas ubiquitin/proteasome system is involved in rapid degradation of proteins, autophagy selectively removes protein aggregates and damaged organelles. Failure of these highly adjusted proteolytic systems to maintain basal turnover leads to altered cellular homeostasis. During evolution, certain viruses have developed mechanisms to exploit their functions to facilitate their own replication, prevent viral clearance and promote the outcome of infection. In this article, we summarize the current opinion on adenoviruses (Ad) and molecular host cell targets, extending on recent evidences for protein degradation pathways in infected cells. We describe recently identified connections between Ad-mediated proteolysis and viral replication with main emphasis on the function of certain Ad proteins.

Selective autophagy in Drosophila.

Autophagy is an evolutionarily conserved process of cellular self-eating and is a major pathway for degradation of cytoplasmic material by the lysosomal machinery. Autophagy functions as a cellular response in nutrient starvation, but it is also associated with the removal of protein aggregates and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Although it was initially believed that autophagy occurs randomly in the cell, during the last years, there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective. Given the important role of autophagy and selective autophagy in several disease-related processes such as neurodegeneration, infections, and tumorigenesis, it is important to understand the molecular mechanisms of selective autophagy, especially at the organismal level. Drosophila is an excellent genetically modifiable model organism exhibiting high conservation in the autophagic machinery. However, the regulation and mechanisms of selective autophagy in Drosophila have been largely unexplored. In this paper, I will present an overview of the current knowledge about selective autophagy in Drosophila.

Loss of autophagy promotes murine acetaminophen hepatotoxicity

Previous reports indicate that mitochondrial dysfunction is essential for the development of liver injury due to acetaminophen. On the other hand, autophagy, which is a major catabolic pathway, plays a critical role in removing protein aggregates, as well as damaged or excess mitochondria in order to maintain intracellular homeostasis. The aim of this study was to clarify if autophagy is linked to liver injury due to acetaminophen. Acetaminophen was administered intraperitoneally to liver-specific Atg7-deficient mice and wild-type mice. A variety of cellular and molecular approaches were used to evaluate the role of autophagy in acetaminophen-induced cell death. Treatment with acetaminophen induced formation of autophagosomes in hepatocytes from wild-type mice, but not in Atg7-deficient mice. Autophagy deficiency enhanced acetaminophen-induced liver injury and activation of caspase-3 and -7 in the liver. Acetaminophen-induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, and JNK activation in hepatocytes were accelerated by autophagy deficiency. The combination of cyclosporin A or JNK inhibitor (SP600125) with acetaminophen blunted both acetaminophen-induced apoptotic and necrotic cell death in autophagy-deficient hepatocytes. Induction of autophagy during acetaminophen treatment plays a pivotal role in the protection against acetaminophen-induced hepatotoxicity through the removal of damaged mitochondria and oxidative stress.

Removal versus fragmentation of amyloid‐forming precursors via membrane filtration

The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥ 100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (< 100 nm) showed no beneficial effect and, in some cases, accelerated insulin fibril formation. This effect may be due to fragmentation of pre-existing aggregates during filtration through small pore membranes, which can increase the number of amyloid-forming precursors. These findings reveal the complexity of removing protein aggregates via filtration and suggest optimal filtration protocols for conducting fibril formation analysis of insulin and similar amyloidogenic proteins.

Removing Protein Aggregates: The Role of Proteolysis in Neurodegeneration

A common characteristic of neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) is the accumulation of protein aggregates. This reflects a severe disturbance of protein homeostasis, the proteostasis. Here, we review the involvement of the two major proteolytic machineries, the ubiquitin proteasome system (UPS) and the autophagy/lysosomal system, in the pathogenesis of neurodegenerative diseases. These proteolytic systems cooperate to maintain the proteostasis, as is indicated by intricate cross talk. In addition, the UPS and autophagy are regulated by stress pathways that are activated by disturbed proteostasis, like the unfolded protein response (UPR). We will specifically discuss how these proteolytic pathways are affected in neurodegenerative diseases. We will show that there is a differential involvement of the UPS and autophagy in different neurodegenerative disorders. In addition, the proteolytic impairment may be primary or secondary to the pathology. These differences have important implications for the design of therapeutic strategies. The opportunities and caveats of targeting the UPS and autophagy/lysosomal system as a therapeutic strategy in neurodegeneration will be discussed.

Stressin' Sestrins take an aging fight

Sestrins (Sesns) are a family of highly conserved stress-responsive proteins, transcriptionally regulated by p53 and forkhead transcription factor that exhibit oxidoreductase activity in vitro and can protect cells from oxidative stress. However, their major biochemical and physiological function does not appear to depend on their redox (reduction and oxidation) activity. Sesns promote activation of adenosine-5′-monophosphate (AMP)-dependent protein kinase in both mammals and flies. Stress-induced Sesn expression results in inhibition of the target of rapamycin complex 1 (TORC1) and the physiological and pathological implications of disrupting the Sesns-TORC1 crosstalk are now being unravelled. Detailing their mechanism of action and exploring their roles in human physiology point to exciting new insights to topics as diverse as stress, cancer, metabolism and aging.

Selective autophagy: ubiquitin-mediated recognition and beyond

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Whereas the ubiquitin-proteasome system is involved in the rapid degradation of proteins, autophagy pathways can selectively remove protein aggregates and damaged or excess organelles. Proteasome-mediated degradation requires previous ubiquitylation of the cargo, which is then recognized by ubiquitin receptors directing it to 26S proteasomes. Although autophagy has long been viewed as a random cytoplasmic degradation system, the involvement of ubiquitin as a specificity factor for selective autophagy is rapidly emerging. Recent evidence also suggests active crosstalk between proteasome-mediated degradation and selective autophagy. Here, we discuss the molecular mechanisms that link autophagy and the proteasome system, as well as the emerging roles of ubiquitin and ubiquitin-binding proteins in selective autophagy. On the basis of the evolutionary history of autophagic ubiquitin receptors, we propose a common origin for metazoan ubiquitin-dependent autophagy and the cytoplasm-to-vacuole targeting pathway of yeast.

Abstract 4150: Exploration of the role of Hsps in exosome derived from prostate cancer cells

Abstract Introduction: Prostate cancer (PCa) is the leading type of cancer diagnosed in men. In 2007 approximately 218, 890 new cases of prostate cancer were reported in the United States (American Cancer Society). Initially the disease is locally confined to the prostate and is hormone or androgen-dependent. It is curable at this stage upon surgical or radiation treatment however, with time, which varies from months to years, many prostate cancers metastasize and even with aggressive hormone ablation treatment approaches, progress to castration resistance (CRPC) and lethality. During early metastasis a response to androgen deprivation therapy (ADT) is usually observed however despite a reduction in androgen levels after ADT, androgen receptor remains active contributing to CRPC progression. Hsp27 is a stress-activated cytoprotective chaperone, up-regulated in CRPC, which predicts a poor prognosis and confers hormone and chemo-resistance in animal models. Targeting Hsp27 using antisense delays time post castration and enhances taxane activity. Recent studies linked cancer with exosome formation; exosomes are 30-100 nm membrane-bound compartment secreted form normal and tumor cells. The mechanisms underlying exosome formation and secretion are not fully understood. Factors such as stage of cancer, cell type and cell cycle could affect the amount and composition of exosomes formed and secreted. Since Hsp27 is up-regulated in CRPC, we hypothesize that Hsp27 can influence exosome formation in androgen independent PCa. Methods-Results: PC3 cells were seeded and grown in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum until 70% confluency. Media was changed to 5% charcoal strip serum DMEM and finally to a serum free media for 72 hours before exosome isolation. Exosomes were purified from the conditioned media using differential centrifugation after remove cell debris and protein aggregates. Further analysis by transmission electron microscopy detected the integrity of purified exosomes. Western blot analysis was also used to probe for different exosome markers including Actin, Tubulin, TSG 101, HSP70 and CD36. In this study, we functionally analyzed the role of Hsp27 in relation to exosome formation from androgen independent prostate cancer cells. Hsp27 was knocked down in PC3 cells using siRNA, and confirmed with western blot analysis. The relative content of Hsp27 in exosomes mirrors and is therefore reflective of that found in cells. Upon Hsp27 knockdown in PC3 cells we can also observe that Hsp27 does not influence exosome formation. Conclusion: Hsp27 chaperone protein is expressed within exosomes, perhaps serving as a novel exosome marker in prostate cancer cell, while Hsp27 knockdown does not affect exosome formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4150.

Abstract A39: A newly defined role of clusterin in the formation of exosomes derived from prostate cancer cells

Abstract Introduction: Molecular chaperones, including heat-shock proteins (Hsps), help cells cope with stress-induced protein misfolding and aggregation and play prominent roles in cellular signaling and transcriptional regulatory networks asscociated with cancer progression and treatment resistance. Clusterin (CLU) is a stress-activated cytoprotective chaperone, up-regulated in Castration Resistant Prostate Cancer (CPRC), that confers hormone and chemo-resistance. Targeting inhibition of CLU using OGX-011 enhances treatment-induced cell death therapeutic response as is currently in clinical trials of men with advanced prostate cancer. Recent studies have linked cancer with exosome formation; exosomes are 30–100 nm membrane-bound compartments secreted from normal and tumor cells. The mechanisms underlying exosome formation and secretion are not fully understood. Factors such as stage of cancer, cell type and cell cycle could affect the amount and composition of exosomes formed and secreted. Since CLU has chaperone activity with a potent ability to inhibit amorphous and fibrillar aggregation of many different proteins, we hypothesize that it can influence exosome formation and release from androgen independent PCa cells. Methods-Results: PC3 cells were seeded and grown in DMEM with 5% FBS until 70% confluency. Media was changed to 5% charcoal strip serum DMEM and finally to a serum free media for 72 hours before exosome isolation. Exosomes were purified from the conditioned media using differential centrifugation after remove cell debris and protein aggregates. Further analysis by transmission electron microscopy detected the integrity of purified exosomes and their concentration. Western blot analysis was also used to probe for different exosome markers including Actin, Tubulin, TSG 101, HSP70 and CD36. In this study, we functionally analyzed the role of Clusterin in relation to exosome formation from PC-3 PCa cells. Clusterin was knocked down in PC3 cells using siRNA, and confirmed with western blot analysis. The content of Clusterin in exosomes after knockdown remains unchanged while, in contrast, cells become Clusterin depleted. Upon Clusterin knockdown in PC3 cells we also observe a dramatic reduction in the amount of exosomes formed. Conclusion: We have found high levels of CLU in exosomes derived from PCa cells. Our data suggests that CLU may serve a role in exosome formation since knocking it down by siRNA dramatically inhibits the quantity of exosomes formed. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A39

Turbidity and Protein Aggregation in Whey Protein Beverages

During storage of heat-treated acidic (pH <or= 4.6) whey protein beverages, formation of protein aggregates can create undesirable turbidity and sedimentation. In this study, we found that a slow protein aggregation process controlled the rate of formation of sediment and turbidity. A heat-treated model beverage containing 12.5 g/L whey protein at a pH of 4 was stored for 6 wk at 3 different storage temperatures and analyzed for turbidity, soluble protein, and protein aggregates. One sample was stored without further treatment and the other sample was filtered to remove protein aggregates formed during heating. This was done to test the hypothesis that aggregates formed during heat treatment served as nuclei for deposition of soluble protein. Turbidity increased and soluble protein decreased as protein aggregates formed during storage. Increasing the storage temperature accelerated this process. The loss of soluble protein during storage was fit to first- and second-order kinetic equations, allowing the prediction of the effect of protein concentration, storage time, and storage temperature on the formation of protein aggregates.