Conformational Diseases Research Articles

Repression of the Heat Shock Response Is a Programmed Event at the Onset of Reproduction

The heat shock response (HSR) is essential for proteostasis and cellular health. In metazoans, agingisassociated with a decline in quality control, thusincreasing the risk for protein conformational disease. Here, we show that in C.elegans, the HSR declines precipitously over a 4hr period in early adulthood coincident with the onset of reproductive maturity. Repression of the HSR occurs due to an increase in H3K27me3 marks at stress gene loci, the timing of which is determined by reduced expression of the H3K27 demethylase jmjd-3.1. This results in a repressed chromatin state that interferes with HSF-1 binding and suppresses transcription initiation in response to stress. The removal of germline stem cells preserves jmjd-3.1 expression, suppresses the accumulation of H3K27me3 at stress gene loci, and maintains the HSR. These findings suggest that competing requirements of the germline and soma dictate organismal stress resistance as animals begin reproduction.

Regulation of protein homeostasis in neurodegenerative diseases: the role of coding and non-coding genes.

Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell’s response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding, trafficking and clearance of proteins, all of which act in an orchestrated manner to ensure proteome stability. The protein quality control system is enhanced by stress response pathways, which take action whenever the proteome is challenged by environmental or physiological stress. Aging, however, damages the proteome, and such proteome damage is thought to be associated with aging-related diseases. In this review, we discuss the different cellular processes that define the protein quality control system and focus on their role in protein conformational diseases. We highlight the power of using small organisms to model neurodegenerative diseases and how these models can be exploited to discover genetic modulators of protein aggregation and toxicity. We also link findings from small model organisms to the situation in higher organisms and describe how some of the genetic modifiers discovered in organisms such as worms are functionally conserved throughout evolution. Finally, we demonstrate that the non-coding genome also plays a role in maintaining protein homeostasis. In all, this review highlights the importance of protein and RNA homeostasis in neurodegenerative diseases.

Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones.

Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases.

Diabetes mellitus as a conformational disease: a novel potential therapeutic approach

Diabetes mellitus as a conformational disease: a novel potential therapeutic approach

The potential of chitosan and its derivatives in prevention and treatment of age-related diseases.

Age-related, diet-related and protein conformational diseases, such as atherosclerosis, diabetes mellitus, cancer, hypercholesterolemia, cardiovascular and neurodegenerative diseases are common in the elderly population. The potential of chitosan, chitooligosaccharides and their derivatives in prevention and treatment of age-related dysfunctions is reviewed and discussed in this paper. The influence of oxidative stress, low density lipoprotein oxidation, increase of tissue stiffness, protein conformational changes, aging-associated chronic inflammation and their pathobiological significance have been considered. The chitosan-based functional food also has been reviewed.

ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3.

The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation.

Localization of mutant ubiquitin in the brain of a transgenic mouse line with proteasomal inhibition and its validation at specific sites in Alzheimer's disease.

Loss of protein quality control by the ubiquitin-proteasome system (UPS) during aging is one of the processes putatively contributing to cellular stress and Alzheimer's disease (AD) pathogenesis. Recently, pooled Genome Wide Association Studies (GWAS), pathway analysis and proteomics identified protein ubiquitination as one of the key modulators of AD. Mutations in ubiquitin B mRNA that result in UBB+1 dose-dependently cause an impaired UPS, subsequent accumulation of UBB+1 and most probably depositions of other aberrant proteins present in plaques and neurofibrillary tangles. We used specific immunohistochemical probes for a comprehensive topographic mapping of the UBB+1 distribution in the brains of transgenic mouse line 3413 overexpressing UBB+1. We also mapped the expression of UBB+1 in brain areas of AD patients selected based upon the distribution of UBB+1 in line 3413. Therefore, we focused on the olfactory bulb, basal ganglia, nucleus basalis of Meynert, inferior colliculus and raphe nuclei. UBB+1 distribution was compared with established probes for pre-tangles and tangles and Aβ plaques. UBB+1 distribution found in line 3413 is partly mirrored in the AD brain. Specifically, nuclei with substantial accumulations of tangle-bearing neurons, such as the nucleus basalis of Meynert and raphe nuclei also present high densities of UBB+1 positive tangles. Line 3413 is useful for studying the contribution of proteasomal dysfunction in AD. The findings are consistent with evidence that areas outside the forebrain are also affected in AD. Line 3413 may also be predictive for other conformational diseases, including related tauopathies and polyglutamine diseases, in which UBB+1 accumulates in their cellular hallmarks.

The biology of proteostasis in aging and disease.

Loss of protein homeostasis (proteostasis) is a common feature of aging and disease that is characterized by the appearance of nonnative protein aggregates in various tissues. Protein aggregation is routinely suppressed by the proteostasis network (PN), a collection of macromolecular machines that operate in diverse ways to maintain proteome integrity across subcellular compartments and between tissues to ensure a healthy life span. Here, we review the composition, function, and organizational properties of the PN in the context of individual cells and entire organisms and discuss the mechanisms by which disruption of the PN, and related stress response pathways, contributes to the initiation and progression of disease. We explore emerging evidence that disease susceptibility arises from early changes in the composition and activity of the PN and propose that a more complete understanding of the temporal and spatial properties of the PN will enhance our ability to develop effective treatments for protein conformational diseases.

Dry Eye: A Protein Conformational Disease

The purpose of this study was to determine whether aqueous-deficient dry eyes (ADDE) is a protein conformational disease. Up to now the therapeutic regimen has been based on empirical results, but these observations may unfold new theranostic approaches for ADDE management. Fifty ADDE patients and 46 healthy volunteers were recruited. Schirmer's test, tear breakup time, tear meniscus height, and fluorescein staining tests were conducted on the subjects. Tear protein for ADDE and control patients was collected and extracted using Schirmer's strip. Protein aggregation was studied by appraisal of average protein size, using dynamic light scattering (DLS), fast performance liquid chromatography (FPLC), and synchronous fluorescence spectroscopy (SFS). Dynamic light scattering data showed a comparatively higher abundance of aggregated proteins in ADDE patients than that in controls. For controls, the size distribution of tear proteins was <50 nm in diameter, whereas the size distribution for ADDE individuals was up to 300 nm in diameter. Fast performance liquid chromatography experiments in native tear proteins exhibited minimal difference in the FPLC profiles for ADDE patients and controls. Denatured tear protein FPLC profiles for patients indicated the presence of protein aggregates which were absent in controls. Our hypothesis was further verified by SFS; lower tryptophan fluorescence in ADDE patients is an indication of oxidative stress, which leads to protein aggregation. Aqueous-deficient dry eyes is likely to be a protein conformational disease. Unlike other conformational diseases where single proteins are involved, this may be a reflection of structural loss for a significant fraction of the tear proteome.

Detection of ubiquitinated huntingtin species in intracellular aggregates.

Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

Tanakan as a multimodal cytoprotective factor in general medicine

The first section of the review provides recent results regarding the mechanism of the action of a standardized extract of ginkgo biloba leaves EGb761 (tanakan), its effects on apoptosis, neuroplasticity, hemorheology, inhibition of the amyloid genesis and inflammatory response it cause, activation of proteasomes in conformational diseases. Current experimental and clinical results suggest that EGb761 (tanakan) is a high-effective cytoprotective agent in cognitive disorders of various genesis, cardiovascular diseases (in the process of rehabilitation and as a medicine capable to reduce the volume of brain damage in stroke). The effect of tanakan on anxiety and depression in patients with cognitive impairment should be considered as clinically significant.

The role of autophagy in liver diseases: mechanisms and potential therapeutic targets.

The role of autophagy in liver diseases: mechanisms and potential therapeutic targets.

Neuronal Serotonin Release Triggers the Heat Shock Response in C. elegans in the Absence of Temperature Increase

Cellular mechanisms aimed at repairing protein damage and maintaining homeostasis, widely understood to be triggered by the damage itself, have recently been shown to be under cell nonautonomous control in the metazoan C. elegans. The heat shock response (HSR) is one such conserved mechanism, activated by cells upon exposure to proteotoxic conditions such as heat. Previously, we had shown that this conserved cytoprotective response is regulated by the thermosensory neuronal circuitry of C. elegans. Here, we investigate the mechanisms and physiological relevance of neuronal control. By combining optogenetic methods with live visualization of the dynamics of the heat shock transcription factor (HSF1), we show that excitation of the AFD thermosensory neurons is sufficient to activate HSF1 in another cell, even in the absence of temperature increase. Excitation of the AFD thermosensory neurons enhances serotonin release. Serotonin release elicited by direct optogenetic stimulation of serotonergic neurons activates HSF1 and upregulates molecular chaperones through the metabotropic serotonin receptor SER-1. Consequently, excitation of serotonergic neurons alone can suppress protein misfolding in C. elegans peripheral tissue. These studies imply that thermosensory activity coupled to serotonergic signaling is sufficient to activate the protective HSR prior to frank proteotoxic damage. The ability of neurosensory release of serotonin to control cellular stress responses and activate HSF1 has powerful implications for the treatment of protein conformation diseases.

Chemical corrector treatment ameliorates increased seizure susceptibility in a mouse model of familial epilepsy.

Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.

Involvement of trace elements in the pathogenesis of prion diseases.

Prion diseases are progressive neurodegenerative diseases that are associated with conformational changes that convert normal cellular prion protein (PrP(C)) into an abnormal pathogenic prion protein (PrP(Sc)). It is widely recognized that prion diseases are forms of transmissible amyloidosis and are considered to be protein-misfolding diseases (conformational diseases), a category that also includes Alzheimer's disease. Trace elements play crucial roles in the conformational change affecting PrP(C), and increasing evidence suggests that PrP(C) is a metal-binding protein that is involved in the homeostasis of Cu, Zn, and Fe. In this article, we review the current understanding of links between trace elements and the conformational change to PrP(Sc), based on our studies using synthetic prion peptides, as well as other new findings. We also focus on PrP(Sc)-induced disruption of Ca homeostasis as a molecular mechanism for neurodegeneration in prion diseases. Possible roles of carnosine (ß-alanyl histidine) as a candidate neuroprotective substance use in prion diseases are also discussed.

Protein misfolding cyclic amplification (PMCA): Current status and future directions

Transmissible spongiform encephalopathies (TSEs) most commonly known as prion diseases are invariably fatal neurological disorders that affect humans and animals. These disorders differ from other neurodegenerative conformational diseases caused by the accumulation in the brain of misfolded proteins, sometimes with amyloid properties, in their ability to infect susceptible species by various routes. While the infectious properties of amyloidogenic proteins, other than misfolded prion protein (PrPTSE), are currently under scrutiny, their potential to transmit from cell to cell, one of the intrinsic properties of the prion, has been recently shown in vitro and in vivo. Over the decades, various cell culture and laboratory animal models have been developed to study TSEs. These assays have been widely used in a variety of applications but showed to be time consuming and entailed elevated costs. Novel economic and fast alternatives became available with the development of in vitro assays that are based on the property of conformationally abnormal PrPTSE to recruit normal cellular PrPC to misfold. These include the cell-free conversion assay, protein misfolding cyclic amplification (PMCA) and quaking induced conversion assay (QuIC), of which the PMCA has been the only technology shown to generate infectious prions. Moreover, it allows indefinite amplification of PrPTSE with strain-specific biochemical and biological properties of the original molecules and under certain conditions may give rise to new spontaneously generated prions. The method also allows addressing the species barrier phenomena and assessing possible risks of animal-to-animal and animal-to-human transmission. Additionally, its unprecedented sensitivity has made possible the detection of as little as one infectious dose of PrPTSE and the biochemical identification of this protein in different tissues and biological fluids, including blood, cerebral spinal fluid (CSF), semen, milk, urine and saliva during the pre-clinical and clinical phases of the disease. The mechanistic similarities between TSEs and other conformational disorders have resulted in the adaptation of the PMCA to the amplification and detection of various amyloidogenic proteins. Here we provide a compelling discussion of the different applications of this technology to the study of TSEs and other neurodegenerative diseases.

Dewetting transition assisted clearance of (NFGAILS) amyloid fibrils from cell membranes by graphene.

Clearance of partially ordered oligomers and monomers deposited on cell membrane surfaces is believed to be an effective route to alleviate many potential protein conformational diseases (PCDs). With large-scale all-atom molecular dynamics simulations, here we show that graphene nanosheets can easily and quickly win a competitive adsorption of human islet amyloid polypeptides (hIAPP22-28) NFGAILS and associated fibrils against cell membrane, due to graphene's unique two-dimensional, highly hydrophobic surface with its all-sp(2) hybrid structure. A nanoscale dewetting transition was observed at the interfacial region between the fibril (originally deposited on the membrane) and the graphene nanosheet, which significantly assisted the adsorption of fibrils onto graphene from the membrane. The π-π stacking interaction between Phe23 and graphene played a crucial role, providing the driving force for the adsorption at the graphene surface. This study renders new insight towards the importance of water during the interactions between amyloid peptides, the phospholipidic membrane, and graphene, which might shed some light on future developments of graphene-based nanomedicine for preventing/curing PCDs like type II diabetes mellitus.

Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets

Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation.We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus’ need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation therapeutics.A key basis for the commonality between viral and neurodegenerative disease aggregation is a broader definition of assembly as more than just simple aggregation, particularly suited for the crowded cytoplasm. The assembly machines are collections of proteins that catalytically accelerate an assembly reaction that would occur spontaneously but too slowly to be relevant in vivo. Being an enzyme complex with a functional allosteric site, appropriated for a non-physiological purpose (e.g. viral infection or conformational disease), these assembly machines present a superior pharmacological target because inhibition of their active site will amplify an effect on their substrate reaction. Here, we present this hypothesis based on recent proof-of-principle studies against Aβ assembly relevant in Alzheimer's disease.

The lower limits for protein stability and foldability in primary hyperoxaluria type I

Mutational effects on protein stability and foldability are important to understand conformational diseases and protein evolution. In this work, we perform a comprehensive investigation on the energetic basis underlying mutational effects on the stability of human alanine:glyoxylate aminotransferase (AGT). We study twenty two variants whose kinetic stabilities span over eleven orders of magnitude and are classified into two groups: i) ten naturally-occurring variants, including the most common mutations causing primary hyperoxaluria type I (PH1); and ii) twelve consensus variants obtained by sequence-alignment statistics. We show that AGT dimer stability determines denaturation rates, and mutations modulate stability by changes in the effective thermodynamic stability, the aggregation propensity of partially/globally unfolded states and subtle energetic changes in the rate-limiting denaturation step. In combination with our previous expression analyses in eukaryotic cells, we propose the existence of two lower limits for AGT stability, one linked to optimal folding efficiency (close to the major allele stability) and the other setting a minimal efficiency compatible with glyoxylate detoxification in vivo (close to the minor allele stability). These lower limits could explain the high prevalence of misfolding as a disease mechanism in PH1 and support the use of pharmacological ligands aimed to increase AGT stability as therapies for this disease.

Structure modeling and dynamics driven mutation and phosphorylation analysis of Beta-amyloid peptides.

The most common characteristics of diverse age-related neurodegenerative diseases are aggregation and accumulation of the misfolded protein in the brain. Alzheimer׳s disease (AD) is one of these protein conformational diseases. Extracellular accumulation of amyloid β (Aβ) is one the neuropathological hallmarks of Alzheimer disease. Various studies have shown that mutation in specific hydrophobic region of Aβ protein inhibit the formation of β sheet, thus aggregation of this protein is stalled. The identification of such mutation in Aβ protein can help us in elucidating the etiology of sporadic Aβ. In our study we have selected three positions: 19ILU, 21ALA and 41ILU in Aβ protein based on their hydrophobic nature and substituted them with PRO ( βSheet breaker). The effects of the substitutions were analysed using molecular dynamics simulation studies. The results validated that the mutations in the specified regions change the hydrophobicity of the protein and the βsheet formation was declined to zero per cent.