RICERCA ITALIANA     

Protein interactomes: unravelling cellular networks in different pathophysiological conditions

Università degli Studi di Napoli "Federico II"

Abstract

Cellular systems depend on multiprotein complexes in which individual proteins assemble into functional modules. Most gene products mediate their function within complex networks of interconnected macromolecules, which have topological and dynamic properties that reflect biological phenomena. Thus, an understanding of biological mechanisms and disease processes demands global analyses of the structure, function and dynamics of the complex networks in which macromolecules function.
The aim of this project is to identify, characterize and functionally describe, using biochemical, molecular biology and proteomic-derived technologies, macromolecular interaction networks of three proteins, which are involved in different physiopathological pathways, to generate mechanistic models that will be implemented into a bioinformatics platform. Each research Units will contribute to the project with their specific know-how and facilities.
The Units I of Prof. Salvatore and the Unit II of Prof Orrù will study the function of two proteins: 1) the putative transcription factor AF4, which is involved in the pathogenesis of human acute lymphoblastic leukemia; and 2) the brain specific glycolytic enzyme, aldolase C, recently reported as a moonlight activity protein with prion-protein binding properties. Both proteins are also involved in the pathogenesis of neurodegenerative diseases.
Unit I will use interaction-detection methods (two-hybrid system, affinity co-immunoprecipitation, GST pull-down) to identify macromolecular complex in cells that normally express the protein of interest. Cell systems will be the human HEK293 and SNKBE, for AF4. Aldolase C expression and function will be evaluated in normal adult mouse brain tissues, in brain tissues from mice affected of neurodegenerative diseases, and in human and mouse neuronal tumor cells (PC12, Neuro2a, and SNKBE). Unit II is the reference center for MS analysis in Naples. This Unit will solve AF4 and aldolase C interactome structure using “functional” proteomic techniques, namely 2D gel electrophoresis and MALDI or Q-TOF MS. Unit II will also use “comparative” proteomics methodology (DIGE) to analyze aldolase C differentially regulated proteins in pathologic versus healthy conditions.
Unit III of Prof. Moratti in Pavia will study the molecular interactome of cardiotoxic and nephrotoxic amyloidogenic proteins. A combination of immunoprecipitation and proteomic techniques will be used to characterize protein complexes, which are the target of the amyloid monoclonal immunoglobulin light chain, in rat cardiomiocytes. Unit III will isolate the complexes using a monoclonal antibody that recognizes a central region of the constant domain of the lambda light chains. Unit III will identify immunoprecipitated proteins by 2D gel electrophoresis and multidimensional LC and MALDI or Q-TOF analysis. Unit II of Prof. Orrù will determine protein expression profile variation of cardiomyocytes exposed to cardiotoxic and non-cardiotoxic amyloidogenic protein, by DIGE.To optimize the proteomic strategies planned in the whole project, Unit II and Unit III will share their expertise in the biomolecular mass spectrometry field.
To interpreter the results, each Unit will benefit from the bioinformatics elaborations, which will be performed by the Unit IV of Prof. Zagari. This Unit will use bioinformatics techniques to identify mutually exclusive interactions between various components of the isolated complexes, and molecular modeling and structural comparison methods to detect common surface patches. Bioinformatics and toponomics will be used to identify relevant interaction partners, which will subsequently be validated in vitro and in vivo.
In summary, the expected results of the Project are: 1) to isolate and unravel the structure of the transcriptional regulatory complex containing AF4, and hence the identification new signaling pathways that are deregulated in human leukemia and in neurodegenerative diseases; 2) to clarify the moonlight function of aldolase C in the brain and its links to known or unknown pathogenic mechanisms; 3) to identify interactors of the toxic amyloid oligomeric light chains in heart and kidney, to design a molecular therapy. <<<

Principal Investigator

Francesco Salvatore Università degli Studi di NAPOLI "Federico II"

Research Objectives

The aim of this project is to identify, characterize and functionally describe, using proteomic-derived technologies, macromolecular interaction networks of three proteins, which are involved in different physiological and pathological pathways, to generate mechanistic models that will be implemented into a bioinformatics platform.
Using high-throughput interaction-detection approaches, namely, yeast two-hybrid systems, affinity co-immunoprecipitation, GST pull-down, functional and comparative proteomic analysis, and bioinformatics tools we will determine the function of: 1) the putative transcription factor AF4 which is involved in the pathogenesis of human leukemia and neurodegenerative diseases; 2) the brain specific glycolytic enzyme, aldolase C, recently reported as a “moonlight” activity protein with prion-protein binding properties; and 3) immunoglobulin light chains in systemic amyloidosis pathogenesis.
Each research unit has specific know-how and facilities related to the issues addressed in the project.
A common feature of AF4 and aldolase C is that they are also specifically expressed in Purkinje cells in the brain. AF4 is mutated in the “robotic mouse”, a novel model of autosomal dominant cerebellar ataxia. The mutation, which lies in a highly conserved region in members of the ALF family, significantly reduces the binding affinity of AF4 to the E3 ubiquitin-ligase Siah-1a, which has been isolated with Siah-2 as interacting proteins in the brain. This process leads to a much slower turnover of mutant AF4 by the ubiquitin-proteasome pathway and consequently to its abnormal accumulation in the robotic mouse. Importantly, conservation of the Siah-binding domain of AF4 in all other family members demonstrates that Siah-mediated proteasomal degradation regulates the levels and thus the transcriptional regulatory function of the ALF family. Therefore, deregulation of the AF4 turnover is probably responsible for the oncogenic potential of the leukemic AF4-MLL chimeras, which lack of the AF4 proteasome-binding region, and for neurodegeneration, in the mouse model over-expressing AF4.
We will carry out experiments to identify potential AF4 partners and verify if they belong to the transcriptional regulatory complex that contains AF4. Starting from the identification of the regulatory complex components, the aim of this project is to identify AF4 target genes whose altered regulation might provoke leukemia or neurodegeneration.
Aldolase C is not directly involved in any known pathogenic pathway, whereas aldolase A and B deficiency cause severe hemolytic anemia and hereditary fructose intolerance, respectively. Aldolase C is over-expressed in the brain of patients with different neurological disease. Its ability to degrade the neuronal-specific NF-L mRNA suggests that aldolase C participates in the regulation of the complex circuitry of neuronal gene expression. Furthermore, evidence that aldolase C binds prion-proteins suggests that it might be involved in the pathogenesis of neurodegenerative diseases.
In attempt to shed light on the new functional role of aldolase C and its possible involvement in the mechanisms of neuronal function and dysfunction, we will conduct studies aimed at identifying macromolecular interactors of aldolase C in normal adult mouse brain. Furthermore, we will evaluate, in brain of mice affected of neurodegenerative diseases, gene products whose levels vary in response to aldolase C over-expression. Identification of such proteins will provide further information about the ‘moonlight’ function of aldolase C and its hypothetic role in the pathogenesis of neurodegeneration.
Lastly, we aim to characterize the effect of amyloid protein aggregates in the heart. Actually, “oligomers” or “protofibrils” are believed to be toxic to neighboring cells and to induce cell death by apoptosis. How oligomers exert their toxic effects and whether cellular lesions result from these toxic effects is of enormous practical importance in the context of developing therapeutic strategies for systemic amyloidosis. Because a correlation between amyloid protein aggregates and cytotoxic effects on target tissues has not yet been established, a cellular model representative of the peculiar pathological features occurring in amyloid disease is of extremely important. We aim to identify protein targets of amylodogenic light chains in cardiomyocytes in order to predict possible binding sites and functional implications.
In all cases, we will use co-immunoprecipitation techniques to isolate multi-protein complexes. In the AF4 study, we will isolate multi-protein complexes to verify the role of transcription factor hypothesized for AF4. Moreover, AF4 cDNA, transfected in specific cell lines, will be used as bait to fish out its specific partners in vivo. The use of tagged recombinant proteins makes it easy to co-immunoprecipitate AF4 and all direct or indirect interactors.
The aldolase C studies will aim at identifying aldolase C interactome in adult mouse cerebellum extracts. Again, will use co-immunoprecipitation to isolate multi-protein complexes and an aldolase C antibody directed against the endogenous protein. Complexes will be isolated by micro-spheres covered by protein A. The latter strategy will also be used to characterize the amyloid light chain interactome in rat cardiomyocytes.
We will use functional proteomic analysis to identify the components of each isolated protein complex. In general, immunoprecipitated proteins will be separated by mono- or bidimensional SDS-PAGE or multidimensional chromatography. Complex constituents will be identified by MALDI/MS or MALDI/TOF.
We will also determine, in specific cell systems, the qualitative and/or quantitative effects of aldolase C over-expression or amyloid light chain exposure, respectively. Therefore, differential pathophysiological conditions will be analyzed in vivo, and the resulting variations will be visualized with the DIGE-based comparative proteomic strategy.
Each unit will have access to bioinformatics data to interpreter the results. The ‘in silico’ platform serves to identify mutually exclusive interactions between the various components of the complexes based on a combination of bioinformatics techniques, structural modeling and network analysis tools. Bioinformatics analysis identifies a large fraction of exclusive interactions in a test data set; molecular modeling and structural comparison methods detect common surface patches. In this project, the Unit expert in bioinformatics will validate data about macromolecular complexes obtained experimentally by the other Units and use these data to produce molecular models of the protein complex. Bioinformatics will be used to identify relevant interaction partners, which will subsequently be analyzed in vitro and in vivo.
In summary, the expected results of the project are: 1) isolation and unraveling of the structure of the transcriptional regulatory complex containing AF4, and hence the identification of signaling pathways that are deregulated in human leukemia and in neurodegenerative diseases; 2) clarification of the “moonlight” function of aldolase C in the brain and its links to known or unknown pathogenic mechanisms; 3) elucidation of the molecular network involved in the amyloid-mediated cardiotoxicity, in order to design new therapeutic strategies.
Furthermore, the data collected might be translated to the brain and heart proteome interaction network and so shed light on the general complex mechanism underlying the molecular pathogenesis of systemic amyloidosis and neurodegenerative disorders. <<<

First Results

FUNCTIONAL PROTEOMICS OF AF4. The main expected results of this study are:
- identification of AF4 C-terminal domain interactome;
- isolation of the transcriptional regulatory complex containing AF4;
- identification of AF4 target genes, whose altered regulation might provoke either leukemia or neurodegeneration.
- identification of direct interactors that could give information about AF4 function and regulation pathways.
Our experiments will identify potential AF4 partners and bioinformatics will verify if they belong to the transcriptional regulatory complex that contains AF4.
Indeed, we already identified a series of AF4 molecular partners using as bait the N-terminal peptide, which spans the transactivation domain. We identified p-TEFb kinase and 14-3-3 theta as AF4 interactors. 14-3-3 proteins, involved in human cancer and neurological disorders, regulate many cellular processes by binding to phosphorylated sites in diverse target proteins. 14-3-3s themselves are phosphorylated. It is known that phosphorylation regulates the AF4 function (14). Therefore, 14-3-3 binding may variously govern AF4 regulation by i) inducing conformational changes, (ii) physically occluding sequence-specific or structural features, (iii) scaffolding, and (iv) changing cellular localization.
To assess the function of the putative direct interaction between AF4 and 14-3-3 theta, bioinformatics study of single domains of the large AF4 protein (about 1200 residues) will define minimal interacting surfaces, common surface patches, and key residues that, if phosphorylated, mediate this binding. Finally, we aim to develop and therefore suggest methods to validate direct and indirect interactions that might be used to validate the role of a specific interaction in normal and pathologic conditions.
In conclusion, the information about the possible role of AF4 biological complexes deriving from functional proteomic studies will greatly simplify the task of obtaining molecular and functional models for the understanding of leukemia and neurodegenerative disease pathogenesis.
IDENTIFICATION OF ALDOLASE C PROTEOME. Aldolase C is not directly involved in any known pathogenic pathway, whereas aldolase A and B deficiency cause severe hemolytic anemia and hereditary fructose intolerance, respectively. Aldolase C is over-expressed in the brain of patients with different neurological disease. Its ability to bind the neuronal-specific NF-L mRNA suggests that aldolase C participates in the regulation of the complex circuitry of neuronal gene expression. Furthermore, evidence that aldolase C binds prion-proteins suggests that it might be involved in the pathogenesis of neurodegenerative diseases. In attempt to assign a new functional role of aldolase C and to shed light on its possible involvement in the mechanisms of neuronal function and dysfunction, our study aim at identifying macromolecular interactors of aldolase C. Expected results will be the following:
- By yeast two hybrid screen of a mouse cDNA library, we plan to identify direct aldolase C protein partners;
- Functional proteomics of normal and neurodegenerative brain tissues aims to isolate aldolase C complex with all transient interactors that might modulate its activity;
- Differential expression proteomic experiments (DIGE) should assess the role of neuronal transcription/translation regulator hypothesized for aldolase C. We will evaluate, in normal and neurodegenerative brain tissues, gene products whose levels vary in response to aldolase C over-expression;
- Should these strategies identify one or more aldolase C interactors, bioinformatics will exploit binary interactions between the bait and single, specific partners to extract information about common structural domains that might directly interact;
- Furthermore, structural analysis of specific aldolase C molecular partners will extract information useful to design functional validation methods.
Unraveling the aldolase C interactome in normal cerebellum might help to uncover the cellular processes in which aldolase C is involved, whereas definition of aldolase C interactome in pathologic tissues will give insight into the role of this protein in neurodegeneration.
Furthermore, evaluation of the expression profile variation of proteins belonging to the same proteome should highlight cellular processes that are directly or indirectly modulated by aldolase C in healthy samples and define cellular pathways which are altered in the neurodegenerative state.
Efficient integration of disparate data sets represents a key challenge in proteomics and functional genomics. Therefore, because a common feature of AF4 and aldolase C is that they might participate with various mechanisms in the brain function and/or dysfunction, the collected data might be translated in the brain proteome interaction network to shed light on the general complex mechanism underlining the molecular pathogenesis of neurodegenerative disorders and to design new therapeutic tools.
AMYLOIDOGENIC IMMUNOGLOBULIN LC PROTEOME INTERACTOME. The expected results of the study of the molecular interactions between amyloidogenic, cardiotoxic proteins, specifically monoclonal immunoglobulin light chains, and the cardiomyocytes are:
- identification of the cardiomyocytes receptor(s) interacting with amyloidogenic cardiotoxic proteins;
- characterization of the cascade of molecular interactions that follows the binding of light chains to receptor(s) and the identification of the main signalling pathways activated;
- definition of the molecular mechanisms responsible for the reduced cardiomyocytes contractile function and cytotoxicity.
These expected results may significantly contribute to advancing our knowledge on the molecular basis of the cardiotoxicity exerted by the monoclonal amyloidogenic cardiotoxic light chains. The clinical implications would be of great relevance considering that the amyloid cardiomyopathy is the main determinant of survival which in certain patients is dramatically limited to few weeks.
The identification of the putative myocardial receptor(s) will allow the design of targeted drugs capable of inhibiting the interactions with the cardiotoxic light chains with obvious impact on the patients’ survival. Furthermore, the elucidation of the mechanisms leading to cardiomyocyte dysfunction and death will permit the design of novel therapeutic strategies. For instance, if the results of this study will document an important role of the oxidative stress, anti-oxidant drugs could be tested in the clinical setting. The efficacy of these new therapeutic approaches could be evaluated through the monitoring of cardiac biomarkers, such as the natriuretic peptide type B (BNP and NT-proBNP) which we have previously reported to be very sensitive to the cardiomyocyte stress induced by amyloid proteins (Circulation. 2003;107(19):2440-5 - Blood. 2006;107(10):3854-8).
BIOINFORMATICS STUDY OF INTERACTOME. The expected result of the bioinformatics unit in this project will be:
- to validate experimental data obtained from the other Units about AF4, aldolase C and amyloidogenic LC macromolecular complexes;
- to produce molecular models of the protein complexes;
- to discover sequence/structural features suggestive of occurrence of interaction with the target protein,
- to give a rough indication of the location of the predicted binding site
- to discriminate a direct or transient interaction
- to provide indication about other putative partners that will be analyzed for their reliability.
The occurrence of shared sequence/structure patterns in the interactome of the same protein provides a valuable support to the possible biological role of the interaction. Fist results will came from the bioinformatics analysis applied to the complexes ALD_C-PrP and AF4 N-terminal domain_14-3-3 theta, respectively. Successively, other specific complexes will be selected depending on the progress of the project.
Validated, modeled and analyzed complexes hold key information useful to identify functions that will serve as basis for further experimental investigations. This adds a value to the whole project that fosters collaboration among the participants that use efficiently and synergically the computational/bioinformatic tools. Research activities in subsequent phases will be planned in greater details depending on outcome from this task.
In summary, the expected results of the project are: i) the isolation and unraveling of the structure of the transcriptional regulatory complex containing AF4, and hence the identification new signaling pathways that are deregulated in human leukemia and in neurodegenerative diseases; ii) clarification of the moonlight function of aldolase C in the brain and its links to known or unknown pathogenic mechanisms; iii) identification of interactions of the immunoglobulin light chains with protein targets in cardiomyocytes. Extensive bioinformatics and computational support is a key element to cope with the experimental data on protein interactions obtained using the described technologies. <<<

Timescale

24 months

National and international background

Cellular systems depend on multi-protein complexes in which individual proteins assemble into functional modules (1,2). Most gene products mediate their function within complex networks of interconnected macromolecules, which have topological and dynamic properties that reflect biological mechanisms as well as disease processes. (3,4).
To understand the interaction network in which macromolecules function, high-throughput interaction detection approaches, such as yeast two-hybrid systems (5,6), proteomic analysis (7,8), and ‘in silico' interaction predictions (9-14), have been developed. In particular, proteomic analysis may potentially look for all the major proteins involved in specific pathways and identify differences in protein expression patterns in different cellular states.
To obtain information about biological activities of individual proteins, “functional proteomics” analyzes spatial and temporal properties of molecular networks and fluxes in living cells. In the cell, many processes are governed not only by the relative abundance of proteins but also by rapid and transient regulation of the activity, association and localization of proteins and protein complexes. Proteomics-based approaches were crucial in identifying interacting proteins in large, stable complexes (15). The simplest strategy to identify in vivo proteins that, even transiently, interact with a chosen protein target entails the use of commercially available protein expression systems to generate recombinant protein fused to various tag-epitopes (i.e., GST, FLAG, etc.) (16). The recombinant tagged protein serves as bait to fish its specific partners out from total cellular extracts. Protein components specifically bound in a complex to the bait are co-eluted, separated by electrophoresis and identified by MS techniques (17,18). This strategy fractionates and identifies several interacting proteins of a large complex in a single experiment, and can be used to create large-scale protein interaction maps in living cells. Moreover, the functional proteomic approach amplifies the binary (or direct) interaction network in a complex interactome that includes secondary (or indirect) interactions, thereby giving insight into specific cellular machine processes.
In “expression proteomics”, differential gel electrophoresis (DIGE) technology compares the protein expression profile of a specific cell line or tissue in two different physiopathological states (15,19). It successfully couples the 2D gel high resolutive power with the sensitivity of fluorescent dyes. Biologic extracts separately labeled with two different dyes are mixed and analyzed on the same 2D gel. Superimposition of images reveals differentially expressed proteins. Software-based differential analysis of a singe gel reduces the experimental variability of other quantitative methods. Therefore, the whole system is the most reproducible procedure for quantitative analysis of complex protein systems.
Extensive bioinformatics analysis is a key element to cope with the massive increase in experimental data about protein interactions obtained with these novel technologies (20). Efficient integration of disparate data sets led to topological descriptions of the overall interactome and to local modeling of the complexes and their constituent proteins, which are rich source of information for a more complete functional characterization of the cell. Several promising methods have been successfully applied to this field. For instance, some methods predicted uncharacterized proteins based on interacting partners (21,22); others analyzed the stable topological properties of interaction networks (23); or revealed that the connectivity of well-conserved proteins in the network is negatively correlated with their rate of evolution (24). Once an interaction between two proteins have been detected and validated, state-of-the-art modeling methods can be used to model the molecular details of the complexes. If we know, or are able to predict, with some reliability that two proteins form a complex, we can detect where they interact, i.e. the reciprocal residues involved in the interaction.
The present project has been planned to use these tools to study structural interactomes of three proteins to clarify their role in physiopathological biological systems.
1. AF4 is involved in human acute leukemias (25-28). It belongs to the ALF protein family. ALF proteins probably act as transcription factors, because they activate transcription in an in vitro reporter system. AF4 plays an important role in B and T lymphopoiesis and it seems to have oncogenic properties (29,30).
Robotic mouse is a new model of autosomal dominant cerebellar ataxia that develops adult-onset Purkinje cell loss; it leads the causative mutation in a highly conserved region of AF4, which is also specifically expressed in Purkinje cells in the cerebellum (31). The causative mutation markedly slows the proteasome-mediated degradation of mutant that abnormally accumulates in the cell (31).
AF4 directly interacts with the p-TEFb kinase, which positively regulates transcription elongation through phosphorylation of RNAPolII C-term (32). AF4 associates with p-TEFb and stimulates its kinase activity; p-TEFb phosphorylates AF4 and down-regulates its transactivation activity (32). Therefore, clarification of mechanisms that regulate the AF4 turnover and identification of its target genes would provide important insights into the understanding of the role of AF4 in neurodegeneration and leukemia and therefore to identify potential therapeutic targets.
2. Moonlight activities are a characteristic of single proteins that have multiple functions (33-35). The glycolytic pathway enzymes exemplify the latter possibility because they may also display RNA-binding properties (36). Aldolase is a ubiquitous enzyme catalyzing the reversible aldol cleavage of fructose 1,6-bisphosphate in the glycolysis and gluconeogenesis and fructose 1-phosphate in the dietary fructose metabolism (37). Vertebrate aldolases exist as three isoforms (A, B, C) with different tissue distributions, which are conserved throughout evolution (37-40). Aldolase C is expressed predominantly in brain. It establishes subsets of neurons during cerebellar development (40) or during differentiation of progenitor cells in the subventricular zones of the developing brain (41). Like neurofilament transcripts, aldolases A and C are expressed early in embryonic development, are up-regulated during postnatal life, and are also differentially expressed in complementary cell types (42-44). In the adult mammalian brain, Aldolase C mRNA expression is localized in well-delimitated areas, i.e., cerebellum, hippocampus, medulla (45). In the cerebellum, aldolase C mRNA and protein are expressed in the Purkinje cells in a stripe-like distribution called "zebrin" (40, 46-48). This variable, stripe-like distribution of aldolase C in different brain regions also suggests that aldolase C protein could play other roles in addition to its glycolytic function.
Recent data identified aldolase C as a prion-protein binding protein (49,50). The cellular prion-protein (PrPC) is constitutively expressed on the plasma membrane of neuronal cells (51-53). As aldolase C is expressed predominantly in Purkinje cells, interaction with PrPC reported in normal cerebellum may be required to maintain the long-term survival of these cells (50).
The recent report that Aldolase C regulates expression levels of neurofilament (NF-L) through its interaction with NF-L mRNA, and the severe motor neuron degeneration elicited by a mutation in the 3’UTR of the NF-L mRNA also suggest that aldolase C might participate to the regulation of the complex neuronal gene expression circuitry (54-56). Identification of aldolase C macromolecular interactors might open new perspectives for understanding the new aldolase C functional roles and its involvement in the mechanisms of neuronal function and dysfunction.
3. Systemic amyloidoses (SA) belong to the wide group of "protein misfolding diseases" characterized by deposition in many tissues and organs of a mainly extracellular, pathological substance called amyloid. The amyloid is constituted by insoluble fibrils with beta-sheet secondary structure formed by one of more than 25 normally soluble proteins (57,58). For the generation of aggregation-prone and thereby amyloidogenic protein species, structural modifications, such as mutations, aberrant cleavage or glycosylation, may have important pathogenic implications. At least 12 different proteins may cause specific forms of systemic amyloidosis in human. In all instances, the mother protein is a plasma protein which is transported at the deposition sites where it forms insoluble fibrils. The distribution of deposits in tissues varies strongly between different biochemical forms. Also within the distinct types, there is a wide heterogeneity. The reasons for the heterogeneity, the mechanisms that determine where the amyloid deposits occur, and how amyloid deposits cause cellular and tissue injury, are a matter of vivid debate. The amyloid deposits, e.g. in the heart, may alter the tissue architecture interfering severely with organ function. Various studies focused the hypothesis that abnormal amyloid fibril precursors, known as “oligomers” or “protofibrils”, may induce apoptosis in neighboring cells (59), but exactly how they exert toxic effects is not well understood.
The definition of the molecular species involved in cell toxicity is of great relevance for the development of therapeutic strategies of systemic amyloidosis. Light chain amyloidosis (AL) is the most common type of systemic amyloidosis in industrialized countries. A bone marrow plasma cell clone may produce an amyloidogenic immunoglobulin light chain that causes light chain amyloidosis (AL) (60,61). In AL amyloidosis the heart involvement is by far the most important prognostic factor. The pathogenesis of amyloid heart damage has been investigated in few studies. Investigator of Unit III have reported that cardiac function in AL amyloidosis can rapidly improve following the post-chemotherapy reduction of the circulating amyloidogenic light chain, despite the amount of cardiac amyloid deposits remaining apparently unaltered (62). Liao et al. (63) investigated the effect of LC obtained from patients with non-amyloid disease or from those with non-cardiac, mild cardiac, and severe cardiac AL amyloidosis on the function of isolated mouse heart. Control, non-cardiac, and mild-cardiac LC infusions did not alter ex vivo cardiac function. In contrast, infusion of severe cardiac LC resulted in rapid and marked impairment of ventricular relaxation. These results suggest that amyloid LC proteins may contribute directly to the pathogenesis and the rapid progression of amyloid cardiomyopathy. The same group later reported that physiological concentrations of LC proteins, isolated from patients with amyloid cardiomyopathy, specifically alter cellular redox state in isolated cardiomyocytes (64). The study of the interaction of amyloidogenic LC and cardiac fibroblasts has shown that the cells internalize LC and secrete glycosaminoglycans with enhanced sulfation (65). However, the nature of the molecular species (oligomers, nascent fibrils, mature fibrils) involved in cardiomyocyte toxicity remains elusive as well as the mechanisms of cardiomyocytes dysfunction and death.
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