Abstract Background Cardiac light chain amyloidosis (AL-CA) is a life-threatening disease and the major determinant of prognosis in AL amyloidosis. The management of heart failure (HF) in AL is challenging and gold standard therapies for HF are poorly tolerated or ineffective. Cardiac toxicity of LCs in AL-CA is poorly understood and the comparison of cardiotoxicity of LCs derived from plasma cell dyscrasias (PCDs) such as multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), will improve our understanding of the mechanisms of cardiac damage. Purpose We aimed to 1) genetically identify and biotechnologically produce full-length LCs from patients with AL-CA, MM, MGUS or non-clonal LCs from healthy volunteers (HV), 2) identify LCs' cardiotoxicity and 3) investigate the underlying mechanisms of cardiotoxicity in vitro. Methods Bone marrow derived CD138+ cells from n=7 patients with AL-CA, n=2 patients with MM and n=2 patients with MGUS and peripheral blood mononuclear cells (PBMCs) from n=2 HV were isolated for RNA extraction and characterization of the LC gene family repertoire. At the protein level, LC expression was confirmed by immunoprecipitation in patients' serum followed by top-down proteomics. The overexpressed LC genes in each patient, encoding the full-length clonal LCs were cloned and produced in Shuffle E. coli cells. Two LCs were produced from HV based on the primary protein structure similarity with the patients' LCs. Primary adult ventricular murine cardiomyocytes (pAVMCs) were isolated and exposed at various LC concentrations for evaluation of cell death and investigation of the cardiotoxicity mechanisms via gene and protein expression. LCs folding, oligomerization and amyloidogenic potential were assessed via circular dichroism (CD), SDS page and electron microscopy respectively. Results We successfully identified the LCs responsible for the disease and isolated the respective proteins in all cases (7 AL-CA, 2 MM, 2 MGUS and 3 HV). Despite the similarity of the LCs in conformation as beta-sheet and oligomerization mainly as dimers, 5 out of 7 AL-CA derived LCs led to a different extent of cardiotoxicity in pAVMCs compared to the HV, MM and MGUS derived LCs which did not alter cell viability. Interestingly, these 5 LCs bared the highest amyloidogenic potency. LCs induced different molecular responses leading to cardiomyocyte death. AL-CA proteins κ-type induced apoptosis and overexpression of endoplasmic reticulum stress (ERS) markers while LCs λ-type increased unfolded protein response (UPR) markers and autophagy without inducing apoptosis. All LCs of κ-type including from MM and MGUS patients led to inteleukin-6 mediated inflammation indicating that this mechanism is independent of the observed toxicity. Conclusions AL-CA derived LCs induce cardiotoxicity, which correlates to their amyloidogenic potential via ERS, UPR, autophagy and apoptosis which can be considered targets for cardioprotection. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Hellenic Foundation for Research and Innovation

The steady increase in the number of newly isolated extremophilic microorganisms and the discovery of their enzymes by academic and industrial institutions underlines the enormous potential of extremophiles for application in future biotechnological processes. Enzymes from extremophilic microorganisms offer versatile tools for sustainable developments in a variety of industrial application as they show important environmental benefits due to their biodegradability, specific stability under extreme conditions, improved use of raw materials and decreased amount of waste products. Although major advances have been made in the last decade, our knowledge of the physiology, metabolism, enzymology and genetics of this fascinating group of extremophilic microorganisms and their related enzymes is still limited. In-depth information on the molecular properties of the enzymes and their genes, however, has to be obtained to analyze the structure and function of proteins that are catalytically active around the boiling and freezing points of water and extremes of pH. New techniques, such as genomics, metanogenomics, DNA evolution and gene shuffling, will lead to the production of enzymes that are highly specific for countless industrial applications. Due to the unusual properties of enzymes from extremophiles, they are expected to optimize already existing processes or even develop new sustainable technologies.

ABSTRACT Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB ( Ar ChiA and Ar ChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli . DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of Ar ChiA (881 amino acids [aa]) and Ar ChiB (578 aa). Ar ChiA and Ar ChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of Ar ChiA exhibits 55% identity with a chitodextrinase from Vibrio furnissii . The Ar ChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans . The Ar ChiA chitin-binding domains are homologous to the chitin-binding domain of Ar ChiB. Ar ChiA and Ar ChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of Ar ChiA, Ar ChiB, and chitinase A of Serratia marcescens was studied using differential scanning calorimetry. Ar ChiA and Ar ChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains: a catalytic domain stabilized by Ca2+, a galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results suggest that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low Km and high kcat values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb.

The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387-->Phe, Tyr387-->Trp, Tyr387-->His, Tyr387-->Asn and Tyr387-->Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis.

A gene encoding a highly thermostable extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus was identified. The gene was cloned, sequenced, and expressed in Escherichia coli and Bacillus subtilis. The gene is 1383 base pairs long and encodes a protein of 461 amino acids. The open reading frame of the gene was verified by microsequencing of the recombinant purified enzyme. The deduced amino acid sequence is 25 amino acids longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the membrane. The recombinant alpha-amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to 100 degrees C. alpha-Amylase shows sequence homology to the other known alpha-amylases and belongs to family 13 of glycosyl hydrolases. This extracellular alpha-amylase is not homologous to the subcellular alpha-amylase previously isolated from the same organism.