Deposition Of Insoluble Proteins Research Articles

Emerging Developments in Targeting Proteotoxicity in Neurodegenerative Diseases.

The most common neurodegenerative diseases are Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, frontotemporal lobar degeneration, and the motor neuron diseases, with AD affecting approximately 6% of people aged 65 years and older, and PD affecting approximately 1% of people aged over 60 years. Specific proteins are associated with these neurodegenerative diseases, as determined by both immunohistochemical studies on post-mortem tissue and genetic screening, where protein misfolding and aggregation are key hallmarks. Many of these proteins are shown to misfold and aggregate into soluble non-native oligomers and large insoluble protein deposits (fibrils and plaques), both of which may exert a toxic gain of function. Proteotoxicity has been examined intensively in cell culture and in in vivo models, and clinical trials of methods to attenuate proteotoxicity are relatively new. Therapies to enhance cellular protein quality control mechanisms such as upregulation of chaperones and clearance/degradation pathways, as well as immunotherapies against toxic protein conformations, are being actively pursued. In this article, we summarize the common pathophysiology of neurodegenerative disease, and review therapies in early-phase clinical trials that target the proteotoxic component of several neurodegenerative diseases.

Syntaxin 5 Is Required for the Formation and Clearance of Protein Inclusions during Proteostatic Stress

Spatial sorting to discrete quality control sites in the cell is a process harnessing the toxicity of aberrant proteins. We show that the yeast t-snare phosphoprotein syntaxin5 (Sed5) acts as a key factor in mitigating proteotoxicity and the spatial deposition and clearance of IPOD (insoluble protein deposit) inclusions associates with the disaggregase Hsp104. Sed5 phosphorylation promotes dynamic movement of COPII-associated Hsp104 and boosts disaggregation by favoring anterograde ER-to-Golgi trafficking. Hsp104-associated aggregates co-localize with Sed5 as well as components of the ER, trans Golgi network, and endocytic vesicles, transiently during proteostatic stress, explaining mechanistically how misfolded and aggregated proteins formed at the vicinity of the ER can hitchhike toward vacuolar IPOD sites. Many inclusions become associated with mitochondria in a HOPS/vCLAMP-dependent manner and co-localize with Vps39 (HOPS/vCLAMP) and Vps13, which are proteins providing contacts between vacuole and mitochondria. Both Vps39 and Vps13 are required also for efficient Sed5-dependent clearance of aggregates.

Current diagnosis and treatment of AL amyloidosis in Japan: a nationwide epidemiological survey

Immunoglobulin light chain (AL) amyloidosis is a plasma cell disorder characterized by the deposition of insoluble fibrillary protein derived from misfolded immunoglobulin light chains in several organs, resulting in organ failure and death. However, little information is available about the prevalence and demographic characteristics of AL amyloidosis and the treatment strategy for the condition in Japan. The Amyloidosis Research Committee, Intractable Disease Division, of the Japanese Ministry of Health and Welfare, retrospectively conducted a nationwide survey of Japanese patients with AL amyloidosis who visited affiliated hospitals from January 2012 to December 2014. The number of patients with AL amyloidosis who visited the hospitals during the study period was 3170, and its incidence was estimated to be 4.2 per million person-years in Japan. For the second questionnaire, we collected 741 patients with AL amyloidosis (median age, 65 years; 59% males). The most commonly affected organ was the kidneys, followed by the heart, gastrointestinal tract, and autonomic nervous system. Only 53% of patients were accurately diagnosed by immunohistochemistry using an anti-light chain antibody. Cardiac amyloidosis was diagnosed at later stages. The leading non-transplant regimen was the bortezomib-combined regimen.

Pleural amyloidosis presenting as recurrent bilateral pleural effusions

Amyloidosis is a disease of insoluble protein deposition. It can affect many organ systems but less commonly found in the lung. Pleural effusions secondary to amyloidosis are rare and sparsely reported in the literature. The incidence ranges from 9 to 14 per million person-years. We present a case report of a 77-year-old female with respiratory distress and recurrent bilateral pleural effusions and known intra-abdominal amyloidosis. Initially, we attempted conservative non-operative management using tube thoracostomy drainage with lytic medications. But ultimately, she required decortication with the hope of promoting symphysis of her lung and chest wall to prevent recurrent effusions. Further investigation is warranted in this disease process, as the exact mechanism remains elusive. The same holds true regarding a standardized treatment approach due to the rarity of the disease.

A Case of Gastrointestinal Amyloidosis Presenting Initially as Melena

Amyloidosis is a condition in which abnormal protein folding leads to insoluble fibrillar protein deposition in extracellular tissues, resulting in diverse clinical manifestations. Herein we present a case of amyloidosis diagnosed in a patient whose first clinical symptom was that of gastrointestinal (GI) bleeding. An 86 year old female with hypothyroidism, atrial fibrillation, and chronic obstructive pulmonary disease, was admitted to an outside hospital with shortness of breath, and three months of melena. Laboratory results showed hemoglobin of 7.5 g/dL with baseline hemoglobin one-year prior of 12g/dL. Cardiac MRI there was suggestive of cardiac amyloidosis, and she was subsequently transferred to Westchester Medical Center for a right heart catheterization and biopsy. Repeat laboratory studies were consistent with iron deficiency anemia, and stool studies were positive for occult blood. She was also found to have elevated kappa and lambda free lights chains, with an elevated kappa/lambda ratio. Urine electrophoresis was notable for elevated total protein. Esophagogastroduodenoscopy (EGD) was performed and revealed innumerable fundic gland polyps, with otherwise normal gastric and duodenal mucosa. On colonoscopy, the terminal ileum was coated with black stool, though no source of gastrointestinal bleeding was found. There was a 2 cm lesion in the ascending colon-thought to be a lipoma, a 6 mm polyp in the transverse colon, and a diminutive recto-sigmoid polyp which were all either biopsied or removed. Random biopsies were taken in the rectum. On pathology, the duodenal biopsies, ascending colon ‘lipoma’, transverse colon polyp, and rectal biopsies were positive for amyloid. Clinical manifestations of gastrointestinal amyloidosis range from mild nausea to severe hemorrhage. Endoscopic findings are varied ranging from normal appearing mucosa to that of a fine granular appearance, with erosions, ulcerations or polypoid protrusions. Tissue biopsy is ideally taken from the duodenum or rectum and confirmed with positive Congo red staining or the presence of amyloid fibrils. Treatment should address symptoms and target the underlying disease; e.g., with chemotherapy in those with AL subtype and biologic therapy in those with AA amyloidosis. Heightened awareness that GI bleeding can be the first manifestation of amyloidosis along with timely endoscopic biopsies, will allow for earlier diagnosis and therapeutic treatment.2007_A Figure 1. Normal appearing duodenum, with biopsy positive for amyloid2007_B Figure 2. Ascending colon lipoma

Guarana (Paullinia cupana Mart.) protects against amyloid-β toxicity in Caenorhabditis elegans through heat shock protein response activation

Alzheimer disease (AD) is a progressive neurodegenerative brain disorder that causes significant disruption in normal brain functioning, representing the most common cause of dementia in the elderly. The main hallmark of AD is the presence of amyloid plaques in the brain formed by the deposition of insoluble amyloid protein (Aβ) outside of neurons. Despite intensive investigation of the mechanisms of AD pathogenesis during the past three decades, little has been achieved in terms of effective treatments or ways to prevent the disease. Paullinia cupana, known as guarana, is a plant endemic to the Amazon region in Brazil with several beneficial effects reported, including delayed aging. In this study, we investigated the effects of chronic consumption of guarana ethanolic extract (GEE) on Aβ toxicity using a C. elegans model of AD. We analyzed the behavioral phenotype, oxidative damage and Aβ protein expression in worms treated with GEE. In addition, we investigated the possible role of the heat shock response on the beneficial effects induced by GEE. Overall, our data demonstrate that chronic GEE treatment decreased the formation of Aβ aggregates in C. elegans, preventing the behavioral deficits and the oxidative damage inducible by Aβ expression, due to activation of the heat shock protein (HSP) response. This finding provides a new alternative against amyloidogenic neurodegenerative diseases and other diseases caused by protein accumulation during aging.

Utility of Echocardiogram and Electrocardiogram in the Prediction of TTR Amyloid Diagnosed by Tc-Pyrophosphate SPECT Imaging

Introduction Cardiac transthyretin amyloidosis (ATTR) results from myocardial deposition of insoluble TTR fibril proteins. In contrast to AL cardiac amyloidosis, there are no blood or urine tests to aid in the diagnosis. In clinical practice, ATTR is most commonly suspected when there is unexplained heart failure with preserved ejection fraction (HFpEF), left ventricular hypertrophy (LVH) on transthoracic echocardiogram (TTE), along with low voltage criteria on electrocardiogram (ECG). Definitive diagnosis historically required invasive endomyocardial biopsy, however technetium pyrophosphate (Tc-PYP) scans have recently emerged as a highly useful non-invasive test in the diagnosis of ATTR. We sought to examine the predictive value of multiple ECG and TTE parameters in the diagnosis of Tc-PYP positive ATTR. Methods We performed a retrospective chart review of 34 consecutive patients during January 2017-April 2018 at Scripps Clinic who underwent Tc-PYP scanning for clinically suspected ATTR. Patients were then separated into Tc-PYP positive and negative groups, and ECG and TTE obtained closest to the scan were reviewed. Statistical significance was determined by Mann-Whitney U and Fischer Exact test. Results Twenty patients had Tc-PYP positive scans versus 11 patients with Tc-PYP negative scans. Three patients with “equivocal” reads were excluded from the analysis. Overall no ECG parameters were significantly predictive of Tc-PYP+ scans. Rates of “low voltage” ECGs were similar between both groups (3/11 vs. 1/8, p-NS), and while there was a trend towards lower occurrence of sinus rhythm (5/20 vs. 5/11, p-NS) and increased ventricular-paced rhythms in patients with Tc-PYP+ scans (9/20 vs. 3/11, p-NS), these were not statistically significant. Comparing TTE variables, we found that Tc-PYP+ patients had significantly higher LV mass index values (171 vs. 123, p = 0.026) compared to Tc-PYP- patients. Additional TTE parameters (listed in Table) did not demonstrate any statistical significance. Conclusion In this single center study, we demonstrated that routinely used ECG and TTE parameters in clinical practice such as low voltage ECG and Tissue Doppler velocities lack sensitivity in the prediction of TTR Amyloid by Tc-PYP scans. Of various echocardiographic parameters, only LV mass index was statistically significant in predicting Tc-PYP positive scans. With promising new therapies on the horizon, and the emergence of Tc-PYP SPECT imaging as a highly specific noninvasive imaging modality for the diagnosis of ATTR, clinicians should have a high index of suspicion and should not stop with a TTE or ECG as both modalities lack sensitivity.

The Insoluble Protein Deposit (IPOD) in Yeast.

The appearance of protein aggregates is a hallmark of several pathologies including many neurodegenerative diseases. Mounting evidence suggests that the accumulation of misfolded proteins into inclusions is a secondary line of defense when the extent of protein misfolding exceeds the capacity of the Protein Quality Control System, which mediates refolding or degradation of misfolded species. Such exhaustion can occur during severe proteotoxic stress, the excessive occurrence of aggregation prone protein species, e.g., amyloids, or during ageing. However, the machinery that mediates recognition, recruitment and deposition of different types of misfolded proteins into specific deposition sites is only poorly understood. Since emerging principles of aggregate deposition appear evolutionarily conserved, yeast represents a powerful model to study basic mechanisms of recognition of different types of misfolded proteins, their recruitment to the respective deposition site and the molecular organization at the corresponding site. Yeast possesses at least three different aggregate deposition sites, one of which is a major deposition site for amyloid aggregates termed Insoluble PrOtein Deposit (IPOD). Due to the link between neurodegenerative disease and accumulation of amyloid aggregates, the IPOD is of particular interest when we aim to identify the molecular mechanisms that cells have evolved to counteract toxicity associated with the occurrence of amyloid aggregates. Here, we will review what is known about IPOD composition and the mechanisms of recognition and recruitment of amyloid aggregates to this site in yeast. Finally, we will briefly discuss the possible physiological role of aggregate deposition at the IPOD.

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrPSc) that occur throughout the brain. Formation of synthetic or in vitro PrPSc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrPSc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrPc (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrPc monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (1H–15N) studies revealed that, in comparison to monomeric PrPc, the oligomer has intense amide peak signals in the N-terminal (residues 90–114) and C-terminal regions (residues 226–231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrPc monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.

Reconstitution of Autophagy Improves Vascular Reactivity in Spontaneously Hypertensive Rats

The accumulation of misfolded‐aggregated proteins is linked to an increasing number of degenerative diseases, including hypertension. Although protein damage and misfolding are an inevitable consequence of normal cellular function, multiple proteostasis mechanisms are devoted to the repair or clearance of damaged proteins in order to prevent proteotoxicity. Autophagy is the constitutive and basally active recycling mechanism responsible for protein quality control. Previously, our data has suggested decreased autophagy in conduit and resistance arteries from spontaneously hypertensive rats (SHR); however, direct measurement of autophagic activity, as well as the proteotoxic consequences of undegraded, misfolded protein has not been performed. Furthermore, the effects of reconstituting autophagic activity on vascular reactivity are currently unknown. We hypothesized that mesenteric resistance arteries (MRA) from adult (14 weeks old) SHR would have decreased autophagic activity in lysosomes extracted from arteries and this would be associated with increased misfolded protein oligomers. Therefore, we further hypothesized that upregulation of autophagy via mammalian target of rapamycin (mTOR) inhibition would decrease contraction and improve relaxation in isolated MRA from SHR mounted on wire myographs. We observed that the activity of acid phosphatase, the major acid hydrolase in lysosomes, from arterial lysosomes (pooled mesenteric arteries and aorta) was decreased in SHR (μmol/min, Wistar: 0.51±0.06 vs. SHR: 0.24±0.02, p<0.05). This diminished arterial autophagic activity was observed in conjunction with the increased presence of insoluble protein deposition. Specifically, SHR MRA contained increased fibrillar amyloid oligomers, especially along the endothelium monolayer, as indicated by Congo red staining (Figure 1). When autophagy was reconstituted in isolated MRA via mTOR inhibition with Everolimus (EV; first generation mTOR inhibitor) for 1 hr, we observed that the maximum contraction to phenylephrine was reduced [Emax (mN), Vehicle: 19.8±0.5 vs. EV (0.1 nM): 10.7±0.5 vs. EV (0.1 μM): 8.5±1.0, p<0.05] and the contraction to high concentrations acetylcholine was prevented [LogEC50, Vehicle: −6.9±0.1 vs. EV (0.1 nM): −9.2±0.4 vs. EV (0.1 μM): −8.2±0.1, p<0.05]. Overall, these data reveal that SHR have impaired arterial lysosomal activity and this is associated with the accumulation of misfolded proteins. As a result, reconstituting arterial autophagic activity improved vascular function and could be a novel therapeutic treatment for proteotoxicity in hypertension.Support or Funding InformationNIH (K99GM118885 and P01HL134604).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unilateral localized conjunctival amyloidosis in a patient with a history of contralateral orbit/eyelid lymphoma

Amyloidosis is a disorder characterized by the deposition of insoluble abnormal proteins in the extracellular space. It may occur as a localized lesion or as a systemic disease involving multiple organs and systems. Localized conjunctival amyloidosis is rare and is less frequently associated with systemic involvement. Although amyloidosis itself is a benign lesion involvement of multiple organs and systems is associated with poor prognosis. Diagnosis of amyloidosis is made on biopsy specimens with Congo red staining for the appearance of apple-green birefringence under polarized light microscopy. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is much more sensitive in diagnosing amyloidosis and can determine the type of amyloid deposit. Here we reported a case of conjunctival amyloidosis in a 52 year-old male patient who was presented with left lower eyelid swelling to our medical center. He has a complicated past medical history of anti-phospholipid antibody syndrome, Buerger's disease (thromboangitis obliterans), and small cell lymphoma (SLL) of the right orbit/eyelid. The patient received radiation to the right orbit to treat SLL with therapy completed one and a half years prior to presentation. Physical examination revealed a firm, raised yellowish colored lesion in the left lower conjunctiva. The conjunctival lesion was biopsied, and tissue sections were examined with Congo red stains and LC-MS/MS analysis. The biopsy showed amyloid deposits without evidence of malignancy, and the type of proteins in the deposit was immunoglobulin light chain (AL) of kappa type. A complete work up was taken for possible systemic involvement of amyloidosis and results were all negative. To our knowledge, this is the first case of localized conjunctival amyloidosis with a history of contralateral orbit/eyelid SLL.

Small Bowel Amyloidosis.

The goal of this paper is to review the literature on small bowel amyloidosis. Our review focuses on the underlying etiology, histopathology, clinical features, endoscopic and radiologic findings, and the mainstay of management. The latest research shows changing epidemiological trends of different types of amyloidosis. It also reveals a better understanding of its pathophysiology and shows improvement in treatment outcomes. Amyloidosis is a group of diseases of multiple etiologies and clinical presentations. It is characterized by pathological deposition of insoluble fibrillar proteins within various organs leading to disruption of their structure and function. The classification of amyloidosis includes primary, secondary, dialysis-related, senile, and hereditary. Amyloidosis can be systemic or localized. The incidence of AA amyloidosis is declining in frequency. If the gastrointestinal (GI) tract is involved, the small intestine is the most commonly affected site. Overall, outcomes among patients with newly diagnosed amyloidosis have improved. This article focuses on small bowel amyloidosis.

The Involvement of Post-Translational Modifications in Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder recognized as the most common cause of chronic dementia among the ageing population. AD is histopathologically characterized by progressive loss of neurons and deposits of insoluble proteins, primarily composed of amyloid-β pelaques and neurofibrillary tangles (NFTs). Several molecular processes contribute to the formation of AD cellular hallmarks. Among them, post-translational modifications (PTMs) represent an attractive mechanism underlying the formation of covalent bonds between chemical groups/peptides to target proteins, which ultimately result modified in their function. Most of the proteins related to AD undergo PTMs. Several recent studies show that AD-related proteins like APP, Aβ, tau, BACE1 undergo post-translational modifications. The effect of PTMs contributes to the normal function of cells, although aberrant protein modification, which may depend on many factors, can drive the onset or support the development of AD. Here we will discuss the effect of several PTMs on the functionality of AD-related proteins potentially contributing to the development of AD pathology. We will consider the role of Ubiquitination, Phosphorylation, SUMOylation, Acetylation and Nitrosylation on specific AD-related proteins and, more interestingly, the possible interactions that may occur between such different PTMs.

Primary Amyloidosis Presenting as Hepatomegaly in a Patient With Undiagnosed Multiple Myeloma: A Challenging Case of Pneumococcal Meningitis and Hepatomegaly With Obstructive Jaundice

Inflammation secondary to sepsis can involve multiple organs, including liver. Hepatocellular injury in sepsis results from hypoperfusion and inflammatory cytokines, manifesting as elevated alkaline phosphatase, AST, ALT and bilirubin. However right upper quadrant scans should be performed when in doubt to rule out other life threatening pathologies. We report a case of undiagnosed multiple myeloma and primary amyloidosis with liver and pancreatic involvement, admitted for sepsis secondary to meningitis. A 65-year-old male was brought to hospital after wife found him confused, diaphoretic and nonverbal. He was febrile and somnolent. Lumbar puncture confirmed bacterial meningitis for which he was treated, but later developed jaundice. His liver function tests were consistent with cholestasis. Right upper quadrant (RUQ) ultrasonography (images 1,2) and CT abdomen (image 3) did not show any tumors, stones or strictures but demonstrated massive hepatomegaly. Malignancy work-up was initiated for hypercalcemia, as PTH, PTHrP and vitamin D levels were normal. Serum protein electrophoresis (SPEP) showed abnormal band suggestive of a paraprotein in gamma region (1.00 g/dL), along with elevation of free Kappa and lambda light chains. Bone marrow biopsy revealed 10% plasma cells. Alkaline phosphatase, AST, ALT, and bilirubin, continue to trend up, peaking at 1521 U/L, 138 U/L, 129 U/L and 4.1 mg/dL (2.9 mg/dL- direct bilirubin) respectively. GGT was elevated at 1364 U/L and CA 19-9 at 3386 U/L. MCRP was negative for biliary or pancreatic duct dilatation. Liver biopsy confirmed primary amyloidosis. Hence patient was started on chemotherapy for IgG kappa multiple myeloma and primary amyloidosis, and had significant improvement in liver function tests and CA 19-9 after first cycle. Amyloidosis is a process of extracellular insoluble protein deposition that commonly involves gastrointestinal tract, with up to 90 percent liver involvement. Jaundice along with elevated alkaline phosphatase and transaminases is seen frequently with liver involvement but jaundice is associated with poor prognosis. Liver alone is almost never the sole organ affected, and in the above-reported case, later on biopsy of the pancreas was also consistent with widespread amyloid deposits. Even though amyloidosis represents a rare cause of cholestatic jaundice, it is important to consider it in differentials due to high mortality with median one-year survival of fifty percent in untreated cases.Figure: Right upper quadrant ultrasound - hepatomegaly.Figure: RUQ ultrasound showing normal CBD.Figure: CT abdomen - hepatomegaly.

Phosphorylation and nuclear transit modulate the balance between normal function and terminal aggregation of the yeast RNA-binding protein Ssd1.

Yeast Ssd1 is an RNA-binding protein that shuttles between the nucleus and cytoplasm. Ssd1 interacts with its target mRNAs initially during transcription by binding through its N-terminal prion-like domain (PLD) to the C-terminal domain of RNA polymerase II. Ssd1 subsequently targets mRNAs acquired in the nucleus either to daughter cells for translation or to stress granules (SGs) and P-bodies (PBs) for mRNA storage or decay. Here we show that PB components assist in the nuclear export of Ssd1and subsequent targeting of Ssd1 to PB sites in the cytoplasm. In the absence of import into the nucleus, Ssd1 fails to associate with PBs in the cytoplasm but rather is targeted to cytosolic insoluble protein deposits (IPODs). The association of Ssd1 either with IPOD sites or with PB/SG requires the PLD, whose activity is differentially regulated by the Ndr/LATS family kinase, Cbk1: phosphorylation suppresses PB/SG association but enhances IPOD formation. This regulation likely accrues from a phosphorylation-sensitive nuclear localization sequence located in the PLD. The results presented here may inform our understanding of aggregate formation by RBP in certain neurological diseases.

Congestive Heart Failure with Preserved Ejection Fraction in a Patient with Light Chain (AL) Amyloidosis and Multiple Myeloma

Patient: Male, 53Final Diagnosis: Cardiac amyloidosisSymptoms: Dyspnea • chest pain • swellingMedication: —Clinical Procedure: Diagnostic approachSpecialty: CardiologyObjective:Challenging differential diagnosisBackground:Amyloidosis is characterized by tissue deposition of insoluble fibrillar proteins and it affects almost every organ; there are many types and the heart can be affected in all of them.Case Report:Our report describes a middle-aged man who presented to the Emergency Department with congestive heart failure. Clinical, electrocardiographic, and echocardiographic findings suggested the presence of an infiltrative disease, so an abdominal fat tissue biopsy was performed. A final diagnosis of systemic amyloidosis with heart involvement was made.Conclusions:This case highlights the importance of combining clinical, electrocardiographic, and echocardiographic information in the diagnosis of complex diseases like amyloidosis with heart involvement.

Effect of position-specific single-point mutations and biophysical characterization of amyloidogenic peptide fragments identified from lattice corneal dystrophy patients.

Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor β-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor β-induced protein (amino acid 611–642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.

Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation.

Mammalian and fungal prions arise de novo; however, the mechanism is poorly understood in molecular terms. One strong possibility is that oxidative damage to the non-prion form of a protein may be an important trigger influencing the formation of its heritable prion conformation. We have examined the oxidative stress-induced formation of the yeast [PSI+] prion, which is the altered conformation of the Sup35 translation termination factor. We used tandem affinity purification (TAP) and mass spectrometry to identify the proteins which associate with Sup35 in a tsa1 tsa2 antioxidant mutant to address the mechanism by which Sup35 forms the [PSI+] prion during oxidative stress conditions. This analysis identified several components of the cortical actin cytoskeleton including the Abp1 actin nucleation promoting factor, and we show that deletion of the ABP1 gene abrogates oxidant-induced [PSI+] prion formation. The frequency of spontaneous [PSI+] prion formation can be increased by overexpression of Sup35 since the excess Sup35 increases the probability of forming prion seeds. In contrast to oxidant-induced [PSI+] prion formation, overexpression-induced [PSI+] prion formation was only modestly affected in an abp1 mutant. Furthermore, treating yeast cells with latrunculin A to disrupt the formation of actin cables and patches abrogated oxidant-induced, but not overexpression-induced [PSI+] prion formation, suggesting a mechanistic difference in prion formation. [PIN+], the prion form of Rnq1, localizes to the IPOD (insoluble protein deposit) and is thought to influence the aggregation of other proteins. We show Sup35 becomes oxidized and aggregates during oxidative stress conditions, but does not co-localize with Rnq1 in an abp1 mutant which may account for the reduced frequency of [PSI+] prion formation.

Role of Protein Quality Control Failure in Alcoholic Hepatitis Pathogenesis.

The mechanisms of protein quality control in hepatocytes in cases of alcoholic hepatitis (AH) including ufmylation, FAT10ylation, metacaspase 1 (Mca1), ERAD (endoplasmic reticulum-associated degradation), JUNQ (juxta nuclear quality control), IPOD (insoluble protein deposit) autophagocytosis, and ER stress are reviewed. The Mallory–Denk body (MDB) formation develops in the hepatocytes in alcoholic hepatitis as a consequence of the failure of these protein quality control mechanisms to remove misfolded and damaged proteins and to prevent MDB aggresome formation within the cytoplasm of hepatocytes. The proteins involved in the quality control pathways are identified, quantitated, and visualized by immunofluorescent antibody staining of liver biopsies from patients with AH. Quantification of the proteins are achieved by measuring the fluorescent intensity using a morphometric system. Ufmylation and FAT10ylation pathways were downregulated, Mca1 pathways were upregulated, autophagocytosis was upregulated, and ER stress PERK (protein kinase RNA-like endoplasmic reticulum kinase) and CHOP (CCAAT/enhancer-binding protein homologous protein) mechanisms were upregulated. In conclusion: Despite the upregulation of several pathways of protein quality control, aggresomes (MDBs) still formed in the hepatocytes in AH. The pathogenesis of AH is due to the failure of protein quality control, which causes balloon-cell change with MDB formation and ER stress.

Regression of cardiac amyloidosis following stem cell transplantation: a comparison between echocardiography and cardiac magnetic resonance imaging in long-term survivors

BackgroundAL amyloidosis and multiple myeloma result in extracellular deposition of insoluble fibrillary protein in tissue and organs. Untreated median survival is very poor, and even worse with cardiac involvement. Chemotherapy and peripheral blood stem cell transplantation (PBSCT) have been shown to dramatically improve survival, with hematologic remission documented. Regression of cardiac changes has previously been shown, as assessed by echocardiography (TTE) and cardiac magnetic resonance imaging (CMR). This study is a comparison of TTE and CMR in long-term survivors of cardiac amyloidosis with regression. ResultsFour long-term survivors with cardiac amyloidosis and regression of cardiac features on TTE were identified. Mean age was 60years and average survival was 139months from the time of diagnosis of cardiac involvement. Statistically significant regression of the cardiac features of cardiac amyloidosis were demonstrated on TTE. In these survivors, post-PBSCT structural assessments were similar between TTE and CMR. Classical strain imaging features of cardiac amyloidosis were only present in 50%. All patients had diffuse, patchy gadolinium enhancement on CMR after PBSCT. ConclusionsTreatment of cardiac amyloidosis with chemotherapy and PBSCT may result in regression of abnormalities on TTE with marked improvement in survival. Post treatment, TTE and CMR structural assessments appear similar. Gadolinium imaging suggests that microscopic residual infiltration persists despite macroscopic regression. Significant cardiac improvements with prolonged survival are seen nonetheless. Multimodality imaging has a vital role in the management of cardiac amyloidosis.