Folding Diseases Research Articles

Polarised Asymmetric Inheritance of Accumulated Protein Damage in Higher Eukaryotes

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny.

Self‐Assembly of Fibers and Fibrils

Too much fiber? Protein fibers play an important role in cellular processes, but are also involved in protein-folding diseases, such as Alzheimer's disease. Modern screening and validation models allow the investigation of the aggregation and deaggregation kinetics of protein fibers (see scheme). These studies can serve as a starting point for the development of new therapeutic strategies.

Protein transport into the endoplasmic reticulum: mechanisms and pathologies

Transport into the endoplasmic reticulum (ER) is the crucial step in the biosynthesis of most secretory proteins and many membrane proteins. The products of the SIL1, SEC62 and SEC63 genes act in concert with the SEC61 complex and the molecular chaperones BiP and GRP170 to transport proteins into the ER. Interestingly, recent genetic work has linked mutations in the human and murine SIL1 genes to neurodegeneration, and mutations in the human SEC63 gene to autosomal dominant polycystic liver disease. Furthermore, mutations in the SEC63 gene and overexpression of the SEC62 gene are associated with various human cancers. Therefore, we suggest that these diseases should be considered to be pathologies of protein transport into the ER rather than protein-folding diseases.

Proline to the rescue

Protein misfolding is now recognized to be a major contributing factor in a number of protein folding diseases, including amyotropic laterial sclerosis, cystic fibrosis, Alzheimer's disease, Parkinson's disease, and a host of many different amyloidosis diseases. Protein aggregation and misfolding reactions are also the bane of protein production and impede pharmaceutical drug development. Understanding the fundamental in vivo factors that control the kinetics of protein misfolding is the crucial aspect involved in developing procedures and strategies to avoid this deleterious side reaction. Elegant in vivo work of Nollen et al. (1) has shown that the elements controlling protein homeostasis such as protein synthesis, energy production in the cell, chaperone-dependent protein folding, protein transport, and protein degradation collectively control intracellular protein aggregation. However, it is also known that protein folding is influenced by the presence of intracellular osmolytes that, in turn, dramatically affect protein stabilities, protein folding rates, and protein aggregation. Thus, it would be highly useful if we could determine how osmolytes directly influence the kinetics of protein folding and aggregation in vivo. In this issue of PNAS, Ignatova and Gierasch (2) accomplish this feat by using their earlier in vivo protein aggregation cellular retinoic acid binding protein (CRABP) 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) model system (3) to monitor in vivo formation of amorphous and fibrillar/amyloid-like aggregation reactions driven by either misfolding or polyglutamine (53htt) aggregation in the presence of high intracellular proline concentrations. Interestingly, they find that the aggregation propensities and kinetics of their particular folding variants of CRABP FlAsH are dramatically altered when proline concentration levels are changed before and during the aggregation reaction (Fig. 1). This system allows these investigators to directly visualize for the first time the in vivo effects of rapid accumulation of an intracellular osmolyte during protein aggregation.

ER stress and apoptosis‐inducing mutations in carbonic anhydrase IV associated with retinitis pigmentosa (RP17) respond to chemical chaperones

We examined two human mutations in the carbonic anhydrase IV (CA IV) gene, which are associated with RP17, an autosomal dominant form of retinitis pigmentosa. CA IV is highly expressed in the choriocapillaris, but not expressed in the retina. The R14W CA IV mutation (Rebello et al., Proc. Natl. Acad. Sci. USA 101:6617–6622, 2004) is located in the signal sequence. The subsequently discovered R219S CA IV mutation is located in the sequence of the mature protein. The biological effects of these mutations were studied in transfected COS cells. Although the signal sequence mutation had only a modest effect on total enzyme production (70% of normal), the R219S mutant protein was completely inactive. Both mutations induced upregulation of BiP, PERK, and CHOP, markers of ER stress. Both suppressed levels of production of other secretory proteins. Both dramatically induced apoptosis as measured by Annexin V binding and TUNEL staining. The markers of ER stress and the number of cells undergoing apoptosis were both greatly reduced by growth in 4-phenylbutyric acid or CA inhibitors, which appeared to act as nonspecific and specific chemical chaperones, respectively. We propose that autosomal dominant RP17 associated with both CA IV mutations is a protein folding disease produced by apoptosis in capillary endothelial cells that nourish the overlying retina. These results suggest that patients with RP17 may respond clinically to treatment with chemical chaperones. This research is supported by NIH grant DK40163 to W.S.S.

Misfolded proinsulin accumulates in expanded pre‐Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice

A missense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupting one of the two interchain disulfide bonds results in intracellular accumulation of misfolded proinsulin. We analyzed the secretory pathway of pancreatic beta cells by electron microscopy and morphometry and identified sites of proinsulin accumulation by quantitative immunogold electron microscopy in this protein-folding disease. In Akita mice beta cells, the volume density of dilated endoplasmic reticulum subdomains was increased by 2.9-fold, resulting in a 1.7-fold increased volume density of the entire rough endoplasmic reticulum. The volume density of pre-Golgi intermediates was increased by 4.9-fold, and that of the Golgi apparatus was increase by 3.4-fold. The relative labeling intensity for proinsulin was 2.1-fold higher in dilated endoplasmic reticulum subdomains and 2.9-fold higher in pre-Golgi intermediates as compared with narrow endoplasmic reticulum, resulting in a significantly different distribution pattern between Akita and control mice beta cells (Chi2= 29.97, P<0.001). The numerical density of insulin secretory granules was equal in Akita and control mice beta cells. However, their volume density and average volume were reduced to 20% and their average diameter to 58% in Akita mice. Together, these data demonstrate that misfolded proinsulin accumulates mainly in pre-Golgi intermediates and to a lesser extent in dilated endoplasmic reticulum subdomains, providing evidence for the importance of pre-Golgi intermediates in a protein folding disease.

Protein degradation and protection against misfolded or damaged proteins.

The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

Endoplasmic reticulum stress is a determinant of retrovirus-induced spongiform neurodegeneration.

FrCas(E) is a mouse retrovirus that causes a fatal noninflammatory spongiform neurodegenerative disease with pathological features strikingly similar to those induced by transmissible spongiform encephalopathy (TSE) agents. Neurovirulence is determined by the sequence of the viral envelope protein, though the specific role of this protein in disease pathogenesis is not known. In the present study, we compared host gene expression in the brain stems of mice infected with either FrCas(E) or the avirulent virus F43, differing from FrCas(E) in the sequence of the envelope gene. Four of the 12 disease-specific transcripts up-regulated during the preclinical period represent responses linked to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Among these genes was CHOP/GADD153, which is induced in response to conditions that perturb endoplasmic reticulum function. In vitro studies with NIH 3T3 cells revealed up-regulation of CHOP as well as BiP, calreticulin, and Grp58/ERp57 in cells infected with FrCas(E) but not with F43. Immunoblot analysis of infected NIH 3T3 cells demonstrated the accumulation of uncleaved envelope precursor protein in FrCas(E)- but not F43-infected cells, consistent with ER retention. These results suggest that retrovirus-induced spongiform neurodegeneration represents a protein-folding disease and thus may provide a useful tool for exploring the causal link between protein misfolding and the cytopathology that it causes.

Highly fluctuating protein structures revealed by variable-pressure nuclear magnetic resonance.

Although our knowledge of basic folded structures of proteins has dramatically improved, the extent of our corresponding knowledge of higher-energy conformers remains extremely slim. The latter information is crucial for advancing our understanding of mechanisms of protein function, folding, and conformational diseases. Direct spectroscopic detection and analysis of structures of higher-energy conformers are limited, particularly under physiological conditions, either because their equilibrium populations are small or because they exist only transiently in the folding process. A new experimental strategy using pressure perturbation in conjunction with multidimensional NMR spectroscopy is being used to overcome this difficulty. A number of rare conformers are detected under pressure for a variety of proteins such as the Ras-binding domain of RalGDS, beta-lactoglobulin, dihydrofolate reductase, ubiquitin, apomyoglobin, p13(MTCP1), and prion, which disclose a rich world of protein structure between basically folded and globally unfolded states. Specific structures suggest that these conformers are designed for function and are closely identical to kinetic intermediates. Detailed structural determination of higher-energy conformers with variable-pressure NMR will extend our knowledge of protein structure and conformational fluctuation over most of the biologically relevant conformational space.

Amyloid fibril proteins

Amyloidosis refers to a group of protein folding diseases. Various innocuous and soluble proteins in physiological conditions polymerize to insoluble amyloid fibrils in several serious diseases, including Alzheimer's disease (AD) and prion diseases. In addition, senile amyloidosis is a form of amyloidosis in which the incidence and severity of amyloid deposition increases with age without any apparent predisposing conditions and it was thought that the amyloidosis was related to some physiological changes which accompany ageing. Although the etiology and pathogenesis of amyloid disease are not fully understood, drastic structural changes of the amyloid proteins from the normal forms to the unique β-sheet fibrils is the most important event in amyloid diseases. The present article introduces the three amyloid diseases, AD, prion diseases and mouse senile amyloidosis in which Aβ, PrP Sc and AApoAII amyloid fibrils deposit respectively. We discuss the nucleation dependent polymerization model as a model that explains the kinetics of fibrillization of these amyloid proteins. Exogenous amyloid fibrils may act as templates (nuclei) and change the conformation of endogenous amyloid protein to polymerize into amyloid fibrils. This hypothesis makes the boundary between transmissible and non-transmissible amyloidosis ambiguous and proposes the common pathogenesis for them.

High Pressure NMR Expands the Protein Structure World.

A protein in solution is a thermodynamic entity, consisting of ensemble of conformers ranging from the lowest-energy folded conformer to the highest-energy unfolded conformer. Under physiological conditions, however, populations of higher energy conformers are small and they are not normally detected by NMR spectroscopy nor by X-ray crystallography. A new type of NMR experiment, utilizing the volume property of a protein in conjunction with pressure, enables structural analysis of semi-stable higher energy conformers, thereby extending the structural analysis essentially to the entire conformational space. The result will provide strong basis for understanding protein function, folding and conformational disease.

Investigating protein conformation-based inheritance and disease in yeast.

Our work supports the hypothesis that a protein can serve as an element of genetic inheritance. This protein-only mechanism of inheritance is propagated in much the same way as hypothesized for the transmission of the protein-only infectious agent in the spongiform encephalopathies; hence these protein factors have been called yeast prions. Our work has focused on [PSI(+)], a dominant cytoplasmically inherited factor that alters translational fidelity. This change in translation is produced by a self-perpetuating change in the conformation of the translation-termination factor, Sup35. Most recently, we have determined that new elements of genetic inheritance can be created by deliberate genetic engineering, opening prospects for new methods of manipulating heredity. We have also uncovered evidence that other previously unknown elements of protein-based inheritance are encoded in the yeast genome. Finally, we have begun to use yeast as a model system for studying human protein folding diseases, such as Huntington's disease. Proteins responsible for some of these diseases have properties uncannily similar to those that produce protein-based mechanisms of inheritance.

Transmissible spongiform encephalopathies, amyloidoses and yeast prions: common threads?

Mammalian transmissible spongiform encephalopathy (prion) and amyloid diseases seem to involve the self-propagation of abnormal fibrillar or sub-fibrillar protein aggregates. Similar processes explain protein-mediated inheritance by yeast prions. Indeed, yeast prions are more surely mediated solely by aberrant protein aggregates than are their mammalian namesakes. Tantalizing parallels make yeast prions attractive models of mammalian protein-folding diseases.

Antagonists to the rescue

Antagonists to the rescue

Fibers of tau fragments, but not full length tau, exhibit a cross beta-structure: implications for the formation of paired helical filaments.

We have used X-ray fiber diffraction to probe the structure of fibers of tau and tau fragments. Fibers of fragments from the microtubule binding domain had a cross beta-structure that closely resembles that reported both for neurofibrillary tangles found in Alzheimer's disease brain and for fibrous lesions from other protein folding diseases. In contrast, fibers of full-length tau had a different, more complex structure. Despite major differences at the molecular level, all fiber types exhibited very similar morphology by electron microscopy. These results have a number of implications for understanding the etiology of Alzheimer's and other tauopathic diseases. The morphology of the peptide fibers suggests that the region in tau corresponding to the peptides plays a critical role in the nucleation of fiber assembly. The dramatically different structure of the full length tau fibers suggests that some region in tau has enough inherent structure to interfere with the formation of cross beta-fibers. Additionally, the similar appearance by electron microscopy of fibrils with varying molecular structure suggests that different molecular arrangements may exist in other samples of fibers formed from tau.

Introduction: Molecular chaperones of the ER: their role in protein folding and genetic disease

Introduction: Molecular chaperones of the ER: their role in protein folding and genetic disease

Protein misfolding and prion diseases

The prion diseases provide an intriguing connection between protein folding and neurodegenerative disease. In this review, I explore that importance of protein folding and misfolding in the prion diseases. Thermodynamic and kinetic models are examined in an effort to understand infectious, inherited and sporadic forms of these diseases. These concepts can be generalized to gain insight into other disorders of protein aggregation and deposition such as Alzheimer’s disease.

Review of the fourth Johns Hopkins Protein Folding Meeting

Review of the fourth Johns Hopkins Protein Folding Meeting

Reactions of denatured proteins with other cellular components to form insoluble aggregates and protection by lactoferrin

Elucidation of the mechanism of formation of insoluble protein aggregates is essential to resolve problems such as protein folding diseases. In this study the effects of various types of biomolecules on the aggregation of denatured proteins were investigated. Denatured α-lactalbumin, an acidic protein, was found to be precipitated by lactoferricin, a basic peptide derived from lactoferrin. Denatured lysozyme, a basic protein, by itself showed aggregation, which was promoted by addition of native α-lactalbumin. Heparin and nucleic acids caused almost instant aggregation of denatured lysozyme. Native lactoferricin was also found to aggregate with heparin or nucleic acids. The results show that denatured/misfolded proteins as well as peptides are highly reactive with other cellular components to form insoluble aggregates and suggest a possible mechanism by which protein folding diseases progress. Most of the above aggregation reactions were inhibited by lactoferrin, which could form soluble complexes with denatured α-lactalbumin, heparin, and nucleic acids.