De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.

design of ACE2 protein decoys to neutralize SARS-CoV-2.

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape.

Light chain amyloidosis induced inflammatory changes in cardiomyocytes and adipose-derived mesenchymal stromal cells.

Light chain (AL) amyloidosis is a progressive, degenerative disease characterized by the misfolding and amyloid deposition of immunoglobulin light chain (LC). The amyloid deposits lead to organ failure and death. Our laboratory is specifically interested in cardiac involvement of AL amyloidosis.

Mechanistic Insights into the Early Events in the Aggregation of Immunoglobulin Light Chains.

Little is known about the mechanism of amyloid assembly in immunoglobulin light chain (AL) amyloidosis, in contrast to other amyloid diseases. Early events in the aggregation pathway are especially important, as these soluble species could be cytotoxic intermediates playing a critical role in the initiation of the amyloid assembly. In this work, we discuss the mechanism of the early events in in vitro fibril formation of immunoglobulin light chain AL-09 and AL-12 (involved in cardiac amyloidosis) and its germline (control) protein κI O18/O8.

Assays for Light Chain Amyloidosis Formation and Cytotoxicity.

Common biophysical techniques like absorption and fluorescence spectroscopy, microscopy, and light scattering studies have been in use to investigate fibril assembly for a long time. However, there is sometimes a lack of consensus from the findings of an individual technique when compared in parallel with the other techniques. In this chapter, we aim to provide a concise compilation of techniques that can effectively be used to obtain a comprehensive representation of the structural, aggregation, and toxicity determinants in immunoglobulin light chain amyloidosis.

Immunoglobulin light chain amyloid aggregation.

Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease. It is characterized by an abnormal proliferation of plasma cells (fully differentiated B cells) producing an excess of monoclonal immunoglobulin light chains that are secreted into circulation, where the light chains misfold, aggregate as amyloid fibrils in target organs, and cause organ dysfunction, organ failure, and death. In this article, we will review the factors that contribute to AL amyloidosis complexity, the findings by our laboratory from the last 16 years and the work from other laboratories on understanding the structural, kinetics, and thermodynamic contributions that drive immunoglobulin light chain-associated amyloidosis.

Mesenchymal stromal cells protect human cardiomyocytes from amyloid fibril damage.

Background Aims: Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins.

Kinetic stability and sequence/structure studies of urine-derived Bence-Jones proteins from multiple myeloma and light chain amyloidosis patients.

It is now accepted that the ability of a protein to form amyloid fibrils could be associated both kinetic and thermodynamic protein folding parameters. A recent study from our laboratory using recombinant full-length (encompassing the variable and constant domain) immunoglobulin light chains found a strong kinetic control of the protein unfolding for these proteins. In this study, we are extending our analysis by using urine-derived Bence Jones proteins (BJPs) from five patients with light chain (AL) amyloidosis and four patients with multiple myeloma (MM).

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation.

Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT.

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

Background: Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death.

Immunoglobulin Light Chains Form an Extensive and Highly Ordered Fibril Involving the N- and C-Termini.

Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (V) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 V. Previous work has demonstrated that the thermodynamic stability of native AL-09 V is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation.

Differences in Protein Concentration Dependence for Nucleation and Elongation in Light Chain Amyloid Formation.

Light chain (AL) amyloidosis is a lethal disease characterized by the deposition of the immunoglobulin light chain into amyloid fibrils, resulting in organ dysfunction and failure. Amyloid fibrils have the ability to self-propagate, recruiting soluble protein into the fibril by a nucleation-polymerization mechanism, characteristic of autocatalytic reactions. Experimental data suggest the existence of a critical concentration for initiation of fibril formation.

Solid-state NMR chemical shift assignments for AL-09 V immunoglobulin light chain fibrils.

Light chain (AL) amyloidosis is a systemic disease characterized by the formation of immunoglobulin light-chain fibrils in critical organs of the body. The light-chain protein AL-09 presents one severe case of cardiac AL amyloidosis, which contains seven mutations in the variable domain (V) relative to its germline counterpart, κI O18/O8 V. Three of these mutations are non-conservative-Y87H, N34I, and K42Q-and previous work has shown that they are responsible for significantly reducing the protein's thermodynamic stability, allowing fibril formation to occur with fast kinetics and across a wide-range of pH conditions.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

Background: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity.

Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity.

Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation in vitro has been described as a nucleation-polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or "seeds" accelerate fibril formation.

Mutations can cause light chains to be too stable or too unstable to form amyloid fibrils.

Light chain (AL) amyloidosis is an incurable human disease, where the amyloid precursor is a misfolding-prone immunoglobulin light-chain. Here, we identify the role of somatic mutations in the structure, stability and in vitro fibril formation for an amyloidogenic AL-12 protein by restoring four nonconservative mutations to their germline (wild-type) sequence. The single restorative mutations do not affect significantly the native structure, the unfolding pathway, and the reversibility of the protein.

Thermodynamic and fibril formation studies of full length immunoglobulin light chain AL-09 and its germline protein using scan rate dependent thermal unfolding.

Light chain (AL) amyloidosis is a fatal disease where monoclonal immunoglobulin light chains deposit as insoluble amyloid fibrils. For many years it has been considered that AL amyloid deposits are formed primarily by the variable domain, while its constant domain has been considered not to be amyloidogenic. However recent studies identify full length (FL) light chains as part of the amyloid deposits.

Differential effects on light chain amyloid formation depend on mutations and type of glycosaminoglycans.

Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units.

A bifunctional non-natural tetrapeptide modulates amyloid-beta peptide aggregation in the presence of Cu(ii).

Amyloid-beta peptide (Aβ) aggregation is one of the hallmarks of Alzheimer's disease (AD), and metal ions such as Cu(ii) have been proposed to play a role in amyloid formation and the onset of this progressive neurodegenerative disorder. This study reports the design and characterization of a novel bifunctional non-natural tetrapeptide, Met-Asp-d-Trp-Aib, that is capable of binding copper, competing with Aβ for Cu(ii), and modulating Aβ aggregation. The study of this tetrapeptide provides further insights into the role of Cu(ii) in the Aβ aggregation pathway, and into the design of compounds with therapeutic potential for Alzheimer's disease.

A molten globule intermediate of the von Willebrand factor A1 domain firmly tethers platelets under shear flow.

Clinical mutations in patients diagnosed with Type 2A von Willebrand disease (VWD) have been identified that break the single disulfide bond linking N- and C-termini in the vWF A1 domain. We have modeled the effect of these mutations on the disulfide-bonded structure of A1 by reducing and carboxy-amidating these cysteines. Solution biophysical studies show that loss of this disulfide bond induces a molten globule conformational state lacking global tertiary structure but retaining residual secondary structure.

Kinetic control in protein folding for light chain amyloidosis and the differential effects of somatic mutations.

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein.

Systemic amyloidoses.

The amyloidoses are a group of protein misfolding diseases in which the precursor protein undergoes a conformational change that triggers the formation of amyloid fibrils in different tissues and organs, causing cell death and organ failure. Amyloidoses can be either localized or systemic. In localized amyloidosis, amyloid deposits form at the site of precursor protein synthesis, whereas in systemic amyloidosis, amyloid deposition occurs distant from the site of precursor protein secretion.

Thermodynamic and kinetic characterization of a germ line human lambda6 light-chain protein: the relation between unfolding and fibrillogenesis.

Proteins encoded by the gene segment 6a of the lambda variable light-chain repertoire are strongly associated with amyloid deposition. 6aJL2 is a model protein constructed with the predicted sequences encoded by the 6a and JL2 germ line genes. In this work, we characterized the urea- and temperature-induced unfolding of 6aJL2.

Thermal unfolding of triosephosphate isomerase from Entamoeba histolytica: dimer dissociation leads to extensive unfolding.

In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area.