Influence Protein Aggregation Research Articles

The role of biomolecular condensates in protein aggregation.

There is an increasing amount of evidence that biomolecular condensates are linked to neurodegenerative diseases associated with protein aggregation, such as Alzheimer's disease and amyotrophic lateral sclerosis, although the mechanisms underlying this link remain elusive. In this Review, we summarize the possible connections between condensates and protein aggregation. We consider both liquid-to-solid transitions of phase-separated proteins and the partitioning of proteins into host condensates. We distinguish five key factors by which the physical and chemical environment of a condensate can influence protein aggregation, and we discuss their relevance in studies of protein aggregation in the presence of biomolecular condensates: increasing the local concentration of proteins, providing a distinct chemical microenvironment, introducing an interface wherein proteins can localize, changing the energy landscape of aggregation pathways, and the presence of chaperones in condensates. Analysing the role of biomolecular condensates in protein aggregation may be essential for a full understanding of amyloid formation and offers a new perspective that can help in developing new therapeutic strategies for the prevention and treatment of neurodegenerative diseases.

In vitro modulation of mTOR and mGlur5 influence α-synuclein accumulation

One of the main hallmarks of Parkinson’s disease (PD) is abnormal alpha-synuclein (α-syn) aggregation which forms the main component of intracellular Lewy body inclusions. This short report used preformed α-syn fibrils, as well as an A53T mutant α-syn adenovirus to mimic conditions of pathological protein aggregation in dopaminergic human derived SH-SY5Y neural cells. Since there is evidence that the mTOR pathway and glutamatergic signaling each influence protein aggregation, we also assessed the impact of the mTOR inhibitor, rapamycin and the mGluR5 allosteric modulator, CTEP. We found that both rapamycin and CTEP induced a significant reduction of α-syn fibrils in SH-SY5Y cells and this effect was associated with a reduction in mTOR signaling and enhancement in autophagic pathway factors. These data support the possibility that CTEP (or rapamycin) might be a useful pharmacological approach to target abnormal α-syn accumulation by promoting intracellular degradation or enhanced clearance.

Integrated multi-omics analysis of Huntington disease identifies pathways that modulate protein aggregation

Huntington disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in the protein huntingtin (HTT). Although the length of the polyglutamine repeat correlates with age at disease onset and severity, psychological, cognitive and behavioral complications point to the existence of disease modifiers. Mitochondrial dysfunction and metabolic deregulation are both associated with the HD but, despite multi-omics characterization of patients and model systems, their mechanisms have remained elusive. Systems analysis of multi-omics data and its validation by using a yeast model could help to elucidate pathways that modulate protein aggregation. Metabolomics analysis of HD patients and of a yeast model of HD was, therefore, carried out. Our analysis showed a considerable overlap of deregulated metabolic pathways. Further, the multi-omics analysis showed deregulated pathways common in human, mice and yeast model systems, and those that are unique to them. The deregulated pathways include metabolic pathways of various amino acids, glutathione metabolism, longevity, autophagy and mitophagy. The addition of certain metabolites as well as gene knockouts targeting the deregulated metabolic and autophagy pathways in the yeast model system showed that these pathways do modulate protein aggregation. Taken together, our results showed that the modulation of deregulated pathways influences protein aggregation in HD, and has implications for progression and prognosis. This article has an associated First Person interview with the first author of the paper.

Emerging roles for G-quadruplexes in proteostasis.

How nucleic acids interact with proteins, and how they affect protein folding, aggregation, and misfolding is a still-evolving area of research. Considerable effort is now focusing on a particular structure of RNA and DNA, G-quadruplexes, and their role in protein homeostasis and disease. In this state-of-the-art review, we track recent reports on how G-quadruplexes influence protein aggregation, proteolysis, phase separation, and protein misfolding diseases, and pose currently unanswered questions in the advance of this scientific field.

Germline influences protein aggregation in somatic tissues

Germline influences protein aggregation in somatic tissues

Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials.

Protein aggregation can affect the quality of protein-based therapeutics. Attempting to unravel factors influencing protein aggregation involves systematic studies. These studies often include sodium azide or similar preservatives in the aggregation buffer. This work shows effects of azide on aggregation of two highly purified reference proteins, both a bovine serum albumin (BSA) as well as a monoclonal antibody (NISTmAb). The proteins were aggregated by thermomechanical stress, consisting of simultaneous heating of the solution with gentle agitation. Protein aggregates were characterized by asymmetric flow field flow fractionation (AF4) with light scattering measurements along with quantification by UV spectroscopy, revealing strong time-dependent generation of aggregated protein and an increase in aggregate molar mass. Gel electrophoresis was used to probe the reversibility of the aggregation and demonstrated complete reversibility for the NISTmAb, but not so for the BSA. Kinetic fitting to a commonly implemented nucleated polymerization model was also employed to provide mechanistic details into the kinetic process. The model suggests that the aggregation of the NISTmAb proceeds via nucleated growth and aggregate-aggregate condensation in a way that is dependent on the concentration (and presence) of the azide anion. This work overall implicates azide preservatives as having demonstrable effects on thermomechanical stress and aggregation of proteins undergoing systematic aggregation and stability studies.

Modulation of Amyloid States by Molecular Chaperones.

Aberrant protein aggregation is a defining feature of most neurodegenerative diseases. During pathological aggregation, key proteins transition from their native state to alternative conformations, which are prone to oligomerize into highly ordered fibrillar states. As part of the cellular quality control machinery, molecular chaperones can intervene at many stages of the aggregation process to inhibit or reverse aberrant protein aggregation or counteract the toxicity associated with amyloid species. Although the action of chaperones is considered cytoprotective, essential housekeeping functions can be hijacked for the propagation and spreading of protein aggregates, suggesting the cellular protein quality control system constitutes a double-edged sword in neurodegeneration. Here, we discuss the various mechanisms used by chaperones to influence protein aggregation into amyloid fibrils to understand how the interplay of these activities produces specific cellular outcomes and to define mechanisms that may be targeted by pharmacological agents for the treatment of neurodegenerative conditions.

Dimensions and Interactions of Large T-Cell Surface Proteins.

The first step of the adaptive immune response involves the interaction of T cells that express T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes expressed by antigen-presenting cells (APCs). Exactly how this leads to activation of the TCR and to downstream signaling is uncertain, however. Recent findings suggest that one of the key events is the exclusion of the large receptor-type tyrosine phosphatase CD45, from close contacts formed at sites of T-cell/APC interaction. If this is true, a full understanding of how close contact formation leads to signaling would require insights into the structures of, and interactions between, large membrane proteins like CD45 and other proteins forming the glycocalyx, such as CD43. Structural insights into the overall dimensions of these proteins using crystallographic methods are hard to obtain, and their conformations on the cell surface are also unknown. Several imaging-based optical microscopy techniques have however been developed for analyzing protein dimensions and orientation on model cell surfaces with nanometer precision. Here we review some of these methods with a focus on the use of hydrodynamic trapping, which relies on liquid flow from a micropipette to move and trap membrane-associated fluorescently labeled molecules. Important insights that have been obtained include (i) how protein flexibility and coverage might affect the effective heights of these molecules, (ii) the height of proteins on the membrane as a key parameter determining how they will distribute in cell-cell contacts, and (iii) how repulsive interactions between the extracellular parts of the proteins influences protein aggregation and distribution.

Huntingtin Aggregation Impairs Autophagy, Leading to Argonaute-2 Accumulation and Global MicroRNA Dysregulation

Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates, resulting in alterations in autophagy. However, the consequences of impaired autophagy for neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation as well as post-mortem material from patients with Huntington's disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNA-induced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together, these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation but also directly contributes to global alterations of intracellular post-transcriptional networks.

Glycosaminoglycans have variable effects on α-synuclein aggregation and differentially affect the activities of the resulting amyloid fibrils

Parkinson's disease is mainly a sporadic disorder in which both environmental and cellular factors play a major role in the initiation of this disease. Glycosaminoglycans (GAG) are integral components of the extracellular matrix and are known to influence amyloid aggregation of several proteins, including α-synuclein (α-Syn). However, the mechanism by which different GAGs and related biological polymers influence protein aggregation and the structure and intercellular spread of these aggregates remains elusive. In this study, we used three different GAGs and related charged polymers to establish their role in α-Syn aggregation and associated biological activities of these aggregates. Heparin, a representative GAG, affected α-Syn aggregation in a concentration-dependent manner, whereas biphasic α-Syn aggregation kinetics was observed in the presence of chondroitin sulfate B. Of note, as indicated by 2D NMR analysis, different GAGs uniquely modulated α-Syn aggregation because of the diversity of their interactions with soluble α-Syn. Moreover, subtle differences in the GAG backbone structure and charge density significantly altered the properties of the resulting amyloid fibrils. Each GAG/polymer facilitated the formation of morphologically and structurally distinct α-Syn amyloids, which not only displayed variable levels of cytotoxicity but also exhibited an altered ability to internalize into cells. Our study supports the role of GAGs as key modulators in α-Syn amyloid formation, and their distinct activities may regulate amyloidogenesis depending on the type of GAG being up- or down-regulated in vivo.

Aggregation and conformational stability evaluation of myoglobin in the presence of ionic surfactant

Sodium lauroyl sarcosinate (SLS) is frequently used for the solubilization of inclusion bodies in vitro due to its structural similarity to lipid plasma membrane. There are many factors that could influence protein aggregation propensity, including overall protein surface charge and hydrophobicity. Here, the aggregation pathway of myoglobin protein was studied under different conditions (pH 3.5 and 7.4) in the presence of varying concentrations of SLS to evaluate the underlying forces dictating protein aggregation. Data obtained from Rayleigh light scattering, ThT binding assay, and far-UV CD indicated that SLS have different effects on the protein depending on its concentration and environmental conditions. In the presence of low concentrations of SLS (0.05–0.1 mM), no aggregation was detected at both pH conditions tested. Whereas, as we reach higher SLS concentrations (0.5–10.0 mM), myoglobin started forming larger-sized aggregates at pH 3.5 and not pH 7.4. These results suggest that electrostatics interactions as well as hydrophobic forces play an important role in SLS-induced myoglobin aggregation.

Identification of process conditions influencing protein aggregation in Chinese hamster ovary cell culture.

Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells. Furthermore, rapid and efficient bioprocess optimization has been performed using design of experiments (DoE) strategies. In this work, we describe a DoE-based approach for the analysis of process parameters and cell culture additives influencing protein aggregation in Chinese hamster ovary (CHO) cell cultures. Important bioprocess variables influencing the aggregation of mAb and host cell proteins were identified in initial screening experiments. Response surface modeling was further applied in order to find optimal conditions for the reduction of protein aggregation during cell culture. It turned out that a temperature-shift to 31 °C, osmolality above 420 mOsm/kg, agitation at 100 rpm and 0.04% (w/v) antifoam significantly reduced the level of aggregates without substantial detrimental effects on cell culture performance in our model system. Finally, the aggregation reducing conditions were verified and applied to another production system using a different bioprocess medium and another CHO cell line producing another mAb. Our results show that protein aggregation can be controlled during cell culture and helps to improve bioprocessing of mAbs, by giving insights into the protein aggregation at its origin in mammalian cell culture.

Huntingtin Aggregation Impairs Autophagy Leading to Argonaute-2 Accumulation and Global MicroRNA Dysregulation

Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates resulting in alterations in autophagy. However, the consequences of impaired autophagy on neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation, as well as post-mortem material from patients with Huntington's disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNA-induced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation, but also directly contributes to global alterations of intracellular posttranscriptional networks.

In This Issue

HomeCirculation ResearchVol. 121, No. 6In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published1 Sep 2017https://doi.org/10.1161/RES.0000000000000174Circulation Research. 2017;121:585High Throughput Screen in Cardiomyocytes (p 604)Download figureDownload PowerPointMcLendon et al identify factors influencing protein aggregation within cardiomyocytes.Accumulations of misfolded proteins within cells can impair cellular functions. Cellular aggregates of misfolded proteins are cleared mainly by the autophagic and proteasomal pathways, and this clearance is particularly important in postmitotic cells, such as those in the brain and heart. Indeed, protein aggregates are a hallmark of certain neurodegenerative disorders as well as heart conditions including desmin-related myopathy (DRM)—a disease characterized by heart and skeletal muscle degeneration. Using a mouse cardiomyocyte model of DRM and a library of silencing RNAs, McLendon and colleagues have now carried out an unbiased genome-wide screen for novel genes that either prevent protein aggregates or promote their clearance. The team identified a number of novel candidates, including the tyrosine kinase, Jak1, whose inhibition reduced protein aggregation by at least 50%. Further analysis of Jak1 in the mouse cells revealed that knock down of the kinase increased proteosomal, but not autophagic, activity. This work not only indicates that Jak1 could be a target for promoting protein clearance in DRM and other protein aggregation diseases, but also provides a list of other protein clearance candidates worthy of consideration.ET-1 Stimulates Vasoconstriction Through Rab11A (p 650)Download figureDownload PowerPointZhai et al determine the intracellular mechanics of endothelin-1 induced vasoconstriction.Controlling contraction and relaxation of arteries involves the careful balancing of opposing vasoconstrictive and dilatory signals, which act upon the smooth muscle cells (SMCs) of the vessel walls. Ion channels that regulate both the influx and intracellular levels of calcium within SMCs control contractility, but how the vasoconstrictive and dilatory signals regulate these channels is largely unknown. To learn more, Zhai and colleagues focused on the BK channels of SMCs. They found that the vasoconstrictor endothelin-1 prevents the trafficking of an essential BK subunit (β1) to the plasma membrane of arterial SMCs, and thus diminishes BK activity. Reduced trafficking of β1 to the cell surface involved endothelin-1 activation of protein kinase C followed by phosphorylation (and inhibition) of the intracellular trafficking factor Rab11a. In contrast, nitric oxide, a potent vasodilator, activated Rab11a and promoted trafficking of β1 to the plasma membrane. The study provides insight into the mechanisms controlling arterial constriction and dilation and reveals potential targets, such as Rab11a, that might be manipulated pharmaceutically to regulate vascular contractility.Ambient Temperature Controls Monocyte Egress From Marrow (p 662)Download figureDownload PowerPointWarm ambient temperatures reduce atherosclerosis in mice, report Williams et al.Epidemiological data shows an association between cold temperatures and adverse cardiovascular events. Yet, cold temperatures also stimulate thermogenesis within brown and beige fat, which is thought to improve weight loss and insulin sensitivity. To gain a better understanding of the effects of ambient temperature on cardiovascular disease, Williams and colleagues housed mice that are prone to atherosclerosis in rooms where the ambient temperatures were 4°C, 22°C, or 30°C. Although total plasma cholesterol levels did not differ between the 3 groups, animals housed at 4°C had the greatest plaque burden, while those housed at 30°C had the lowest. The ambient temperature also inversely correlated with the abundance of monocytes emerging from bone marrow and infiltrating plaques, with animals housed at 30°C exhibiting the lowest levels of both and the lowest numbers of circulating monocytes overall. The team went on to show that monocyte counts in humans were also inversely correlated with seasonal temperature changes. Williams and colleagues argue that while activation of brown and beige fat may instigate beneficial metabolic changes for combating obesity, promoting exposure to cold as a means to achieve this end, could have adverse effects. Previous Back to top Next FiguresReferencesRelatedDetails September 1, 2017Vol 121, Issue 6 Advertisement Article InformationMetrics © 2017 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000174 Originally publishedSeptember 1, 2017 PDF download Advertisement

Effects of Different Lipase Inactivation Treatments on Physicochemical Properties of Naked Oat Globulins

Lipase inactivation is an essential treatment for oat processing, because of the negative effects of lipase on nutrient preservation and storage extension. The effects of different lipase inactivation treatments including hot air roasting, infrared roasting, normal‐pressure steaming, and high‐pressure steaming on the physicochemical properties of oat globulins were investigated. Results showed that normal‐pressure steaming had little effect on solubility of oat globulins; hot air roasting increased foaming capacity of oat globulins but did not change their foaming stability; and all the inactivation treatments increased the surface hydrophobicity and content of total sulfhydryls of oat globulin but decreased exposed sulfhydryl groups. In addition, oat globulin granules from the hot air roasting treatment were distributed more evenly in oat globulin powder compared with the control group. All treatments except normal‐pressure steaming changed the molecular weight of oat globulin subunits, which made the bands of 66,000 and 45,000 disappear from SDS‐PAGE. These results indicated that normal‐pressure steaming was ideal to maintain good solubility of oat globulins, and hot air roasting was ideal to maintain relatively good foaming properties. The treatments changed physicochemical properties of oat globulins by influencing protein aggregation and subunit composition that resulted in different content of sulfhydryl groups and surface hydrophobicity.

Self-Assembling NanoLuc Luciferase Fragments as Probes for Protein Aggregation in Living Cells.

Given the clear role of protein aggregation in human disease, there is a critical need for assays capable of quantifying protein aggregation in living systems. We hypothesized that the inherently low background and biocompatibility of luminescence signal readouts could provide a potential solution to this problem. Herein, we describe a set of self-assembling NanoLuc luciferase (Nluc) fragments that produce a tunable luminescence readout that is dependent upon the solubility of a target protein fused to the N-terminal Nluc fragment. To demonstrate this approach, we employed this assay in bacteria to assess mutations known to disrupt amyloid-beta (Aβ) aggregation as well as disease-relevant mutations associated with familial Alzheimer's diseases. The luminescence signal from these experiments correlates with the reported aggregation potential of these Aβ mutants and reinforces the increased aggregation potential of disease-relevant mutations in Aβ1-42. To further demonstrate the utility of this approach, we show that the effect of small molecule inhibitors on Aβ aggregation can be monitored using this system. In addition, we demonstrate that aggregation assays can be ported into mammalian cells. Taken together, these results indicate that this platform could be used to rapidly screen for mutations that influence protein aggregation as well as inhibitors of protein aggregation. This method offers a novel, genetically encodable luminescence readout of protein aggregation in living cells.

Disease Related Point Mutations and Solution Conditions Determine Fibrillization Behavior of α-Synuclein

In vitro fibrillization of proteins into amyloid fibrils provides critical insights into the factors influencing protein aggregation and has a key role in understanding the molecular basis of several neurodegenerative diseases caused by amyloids. α-Synuclein (αSyn), an intrinsically disordered protein implicated in Parkinson's disease, aggregates readily in vitro into fibrils that exhibit appreciable structural polymorphism. This inherent polymorphism is a major obstacle in elucidating structural features and understanding the fibrillization process. By selecting specific solution conditions we were able to produce morphologically homogeneous fibrils of wt and disease mutant αSyn at the plateau phase of Thioflavin-T (ThT) assays, as evident from atomic force microscopy (AFM) imaging and analyses. Our results indicate that the in vitro aggregation conditions as well as the disease related point mutations of the protein determine the dominant morphology and the maturation behavior of the fibrils produced. Specifically, the morphology of wt αSyn fibrils appears to be dictated by two distinct mechanisms that is competitive growth of different polymorphs during the fibrillization phase followed by structural rearrangements during the process of aging. In contrast, the disease mutant αSyn variants aggregate with faster kinetics and result in fibrils with well defined and stable morphology over time. Additional cross seeding experiments of wt αSyn with disease mutant proteins have shown faithful transmission of the mutant fibril morphologies across two generations. The aggregation into homogeneous fibril populations with mutant-specific morphology is characterized by distinct fibrillization kinetics in ThT assays. Moreover, our experiments indicate differential interaction of ThT with morphologically different αSyn amyloid fibrils.

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.

Direct analysis of mAb aggregates in mammalian cell culture supernatant.

BackgroundProtein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP). Current methods to determine aggregate formation during cell culture include size exclusion chromatography (SEC) with a previous affinity chromatography step in order to remove disturbing cell culture components. The pre-purification step itself can already influence protein aggregation and therefore does not necessarily reflect the real aggregate content present in cell culture. To analyze mAb aggregate formation directly in the supernatant of Chinese hamster ovary (CHO) cell culture, we established a protocol, which allows aggregate quantification using SEC, without a falsifying pre-purification step.ResultsThe use of a 3 μm silica SEC column or a SEC column tailored for mAb aggregate analysis allows the separation of mAb monomer and aggregates from disturbing cell culture components, which enables aggregate determination directly in the supernatant. Antibody aggregate analysis of a mAb-producing CHO DG44 cell line demonstrated the feasibility of the method. Astonishingly, the supernatant of the CHO cells consisted of over 75% mAb dimer and larger oligomers, representing a substantially higher aggregate content than reported in literature so far.ConclusionThis study highlights that aggregate quantification directly in the cell culture supernatant using appropriate SEC columns with suitable mAb aggregate standards is feasible without falsification by previous affinity chromatography. Moreover, our results indicate that aggregate formation should be addressed directly in the cell culture and is not only a problem in DSP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0099-3) contains supplementary material, which is available to authorized users.

The crowd you're in with: Effects of different types of crowding agents on protein aggregation

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein–polymer interactions influence the structure and dynamics of proteins in crowded environments.