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Many professional societies utilize travel awards programs to foster inclusion and facilitate the professional development of underrepresented minority (URM) scientists. All member societies that participate in the Alliance to Catalyze Change for Equity in STEM Success (ACCESS) do so to some degree. Members of this meta-organization recently came together to share their different approaches to URM travel award program assessment. The practices of the Biophysical Society (BPS), one of the ACCESS member societies, is used as a case study to discuss the highlights of our findings. We share and discuss a framework for URM travel award program assessment.

BACKGROUND In vivo mechanisms of amyloid clearance and cardiac tissue damage in cardiac amyloidosis are not well understood. OBJECTIVES We aimed to define and quantify the amyloid plaque proteome in cardiac transthyretin amyloidosis (ATTR) and light chain amyloidosis (AL) and identify associations with patient characteristics and outcomes. METHODS A proteomics approach was used to identify all proteins in cardiac amyloid plaques, and to compare both normal and diseased controls. All proteins identified within amyloid plaques were defined as the expanded proteome; only proteins that were enriched in comparison to normal and disease controls were defined as the amyloid-specific proteome. RESULTS Proteomic data from 292 patients with ATTR and 139 patients with AL cardiac amyloidosis were included; 160 and 161 unique proteins were identified in the expanded proteomes, respectively. In the amyloid-specific proteomes, we identified 28 proteins in ATTR, 19 in AL amyloidosis, with 13 proteins overlapping between ATTR and AL. ATTR was characterized by a higher abundance of complement and contractile proteins and AL by a higher abundance of keratins. We found that the proteome of kappa AL had higher levels of clusterin, a protective chaperone, and lower levels of light chains than lambda despite higher levels of circulating light chains. Hierarchical clustering identified a group of patients with worse survival in ATTR, characterized by high levels of PIK3C3, a protein with a central role in autophagy. CONCLUSIONS Cardiac AL and ATTR have both common and distinct pathogenetic mechanisms of tissue damage. Our findings suggest that autophagy represents a pathway that may be impaired in ATTR and should be further studied.

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

This study evaluates newly diagnosed IgM (6%, n = 75/1174) vs. non-IgM light chain amyloidosis patients. IgM amyloid patients had lower light chains (12.5 vs. 22.5 mg/dL; p 1 organ involvement (31% vs. 44%, p = 0.02) was less common in IgM amyloidosis, while soft tissue and peripheral nerve involvement was more common. t(11;14) was less common (27% vs. 50%, p = 0.008) in IgM amyloidosis. Rates of MYD88L265P and CXCR4WHIM mutation in IgM amyloidosis were 58% (29/50) and 17% (8/46). Diagnosis after hematopathology review in IgM amyloidosis was pure plasma cell neoplasm (PPCN) in 23% (16/70), lymphoplasmacytic neoplasm (LPL) in 63% (44/70) patients, and other (14%). LPL vs. PPCN groups had distinct genetic abnormalities: t(11;14): 0% (0/18) vs. 60% (9/15), p

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. Previous work from our laboratory showed that AL-12 adopts a canonical dimer conformation (like the germline protein), whereas AL-09 presents an altered dimer interface as a result of somatic mutations. Both AL-12 and AL-09 aggregate with similar rates and significantly faster than the germline protein. AL-09 is the only protein in this study that forms stable oligomeric intermediates during the early stages of the aggregation reaction with some structural rearrangements that increase the thioflavin T fluorescence but maintain the same number of monomers in solution. The presence of the restorative mutation AL-09 H87Y changes the kinetics and the aggregation pathway compared to AL-09. The single restorative mutation AL-12 R65S slightly delayed the overall rate of aggregation as compared to AL-12. Collectively, our study provides a comprehensive analysis of species formed during amyloid nucleation in AL amyloidosis, shows a strong dependence between the altered dimer conformation and the formation of stable oligomeric intermediates, and sheds light on the structural features of amyloidogenic intermediates associated with cellular toxicity.

This study evaluated the differences in clinical features of 1077 newly diagnosed AL amyloidosis patients with renal involvement (n = 229, 21%), both cardiac and renal involvement (n = 443, 41%) and cardiac involvement (n = 405, 38%). Significant differences in dFLC (difference in involved and uninvolved light chains) were noted (renal, both, cardiac median: 83, 234 and 349 mg/l, P < 0.001). The proportion of patients with ≥ 10% bone marrow plasma cells (BMPCs) was lowest in renal only patients: 44%, 57%, 64%, respectively, P < 0.001. In a multivariate linear regression model incorporating organ involvement type and BMPCs ≥10%, organ involvement was a significant predictor of dFLC (P < 0.001). Median overall survival (OS) across the three groups was 83 vs. 19 vs. 16 months (P < 0.001) in patients not undergoing transplant and 5-year OS in patients undergoing transplant was 90% vs. 75% vs. 64% (P = 0.007), respectively. In conclusion, renal involvement alone or renal + cardiac involvement in AL amyloidosis is associated with lower circulating light chain burden, which cannot be fully explained by BMPC burden alone. Increased sensitivity of the kidney to light chains, given significant interactions with the renal tubular system and secretion of modified light chain products may play a role in pathogenesis of renal AL amyloidosis and warrants further investigation.

Patients with immunoglobulin M (IgM) light chain (AL) amyloidosis have a distinct clinical presentation compared with those with non-IgM amyloidosis. We hypothesized that differential immunoglobulin light-chain variable region (IGVL) gene usage may explain the differences in organ involvement, because IGVL usage correlates with organ tropism. IGVL usage was evaluated by mass spectrometry of amyloid deposits (IgM, n = 45; non-IgM, n = 391) and differed across the 2 groups. In the λ family, LV2-08 (13% vs 2%; P < .001) and LV2-14 (36% vs 10%; P < .001) usage was more common in IgM vs non-IgM amyloidosis, whereas LV1-44 (0% vs 10%; P = .02) and LV6-57 (2% vs 18%; P = .004) usage was less common. In the κ family, there was a trend toward higher KV4-01 (11% vs 4%; P = .06) usage in IgM amyloidosis. IGVL usage correlated with disease characteristics/organ tropism. LV2-14 (more common in IgM amyloidosis) has historically been associated with peripheral nerve involvement and lower light chain burden, which were more frequent in IgM amyloidosis. LV1-44 (less common in IgM), associated with cardiac involvement, was less frequent in IgM patients. LV6-57 (less common in IgM) is associated with t(11;14), which was less frequent in IgM patients. In conclusion, IGVL gene usage differs in patients with IgM vs non-IgM amyloidosis and may explain the distinct clinical presentation.

Early events in immunoglobulin light chain (AL) amyloid formation are especially important as some early intermediates formed during the aggregation reaction are cytotoxic and play a critical role in the initiation of amyloid assembly. We investigated the early events in in vitro aggregation of cardiac amyloidosis AL proteins at pH 7.4. In this study we make distinctions between general aggregation and amyloid formation. Aggregation is defined by the disappearance of monomers and the detection of sedimentable intermediates we call non-fibrillar macromolecular (NFM) intermediates by transmission electron microscopy (TEM). Amyloid formation is defined by the disappearance of monomers, Thioflavin T fluorescence enhancement, and the presence of fibrils by TEM. All proteins aggregated at very similar rates via the formation of NFM intermediates. The condensed NFM intermediates were composed of non-native monomers. Amyloid formation and amyloid yield was variable among the different proteins. During the stationary phase, all proteins demonstrated different degrees of dissociation. These dissociated species could play a key role in the already complex pathophysiology of AL amyloidosis. The degree of dissociation is inversely proportional to the amyloid yield. Our results highlight the importance and physiological consequences of intermediates/fibril dissociation in AL amyloidosis.

Scientific societies aiming to foster inclusion of scientists from underrepresented (UR) backgrounds among their membership often delegate primary responsibility for this goal to a diversity-focused committee. The National Science Foundation has funded the creation of the Alliance to Catalyze Change for Equity in STEM Success (ACCESS), a meta-organization bringing together representatives from several such STEM society committees to serve as a hub for a growing Community of Practice. Its goal is to coordinate efforts to advance inclusive practices by sharing experiences and making synergistic discoveries about what works. ACCESS has analyzed the approaches by which member societies have sought to ensure inclusivity through selection of annual meeting speakers. Not only do we discuss how inclusive speaker selection fosters better scientific environments for all, but also identify challenges and promising practices for societies striving to maximize inclusivity of speakers in their scientific programming.

Diversity-focused committees continue to play essential roles in the efforts of professional scientific societies to foster inclusion and facilitate the professional development of underrepresented minority (URM) young scientists in their respective scientific disciplines. Until recently, the efforts of these committees have remained independent and disconnected from one another. Funding from the National Science Foundation has allowed several of these committees to come together and form the Alliance to Catalyze Change for Equity in STEM Success, herein referred to as ACCESS. The overall goal of this meta-organization is to create a community in which diversity-focused committees can interact, synergize, share their collective experiences, and have a unified voice on behalf of URM trainees in science, technology, engineering, and mathematics disciplines. In this Essay, we compare and contrast the broad approaches that scientific societies in ACCESS use to implement and assess their travel award programs for URM trainees. We also report a set of recommendations, including both short- and long-term outcomes assessment in populations of interest and specialized programmatic activities coupled to travel award programs.

Diversity-focused committees continue to play essential roles in the efforts of professional scientific societies to foster inclusion and facilitate the professional development of underrepresented minority (URM) young scientists in their respective scientific disciplines. Until recently, the efforts of these committees has remained independent and disconnected from one another. Funding from the National Science Foundation has allowed several of these committees to come together and form the Alliance to Catalyze Change for Equity in STEM Success, herein referred to as ACCESS. The overall goal of this meta-organization is to create a community in which diversity-focused committees can interact, synergize, share their collective experiences, and have a unified voice on behalf of URM trainees in STEM disciplines. In this article, we compare and contrast the broad approaches that scientific societies in ACCESS use to implement and assess their travel award programs for URM trainees. We also report a set of recommendations, including both short- and long-term outcomes assessment in populations of interest and specialized programmatic activities coupled to travel award programs.

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. We have previously shown that the fibrillar aggregates of LC proteins can be cytotoxic and arrest the growth of human RFP-AC16 cardiomyocytes in vitro. We showed that adipose-derived mesenchymal stromal cells (AMSC) can rescue the cardiomyocytes from the fibril-induced growth arrest through contact-dependent mechanisms. In this study, we examined the transcriptome changes of human cardiomyocytes and AMSC in the presence of AL amyloid fibrils. The presence of fibrils causes a 'priming' immune response in AMSC associated with interferon associated genes. Exposure to AL fibrils induced changes in the pathways associated with immune response and extracellular matrix components in cardiomyocytes. We also observed upregulation of innate immune-associated transcripts (chemokines, cytokines, and complement), suggesting that amyloid fibrils initiate an innate immune response on these cells, possibly due to phenotypic transformation. This study corroborates and expands our previous studies and identifies potential new immunologic mechanisms of action for fibril toxicity on human cardiomyocytes and AMSC rescue effect on cardiomyocytes.

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). We observed lower stability in κ proteins compared to λ proteins (for both MM and AL proteins) in agreement with previous studies. The kinetic component of protein stability is not a universal feature of BJPs and the hysteresis observed during refolding reactions could be attributed to the inability of the protein to refold all domains. The most stable proteins exhibited 3-state unfolding transitions. While these proteins do not refold reversibly, partial refolding shows 2-state partial refolding transitions, suggesting that one of the domains (possibly the variable domain) does not refold completely. Sequences were aligned with their respective germlines and the location and nature of the mutations were analyzed. The location of the mutations were analyzed and compared with the stability and amyloidogenic properties for the proteins in this study, increasing our understanding of light chain unfolding and amyloidogenic potential.

Polycystic kidney disease (PKD) involves progressive hepatorenal cyst expansion and fibrosis, frequently leading to end-stage renal disease. Increased vasopressin and cAMP signaling, dysregulated calcium homeostasis, and hypertension play major roles in PKD progression. The guanylyl cyclase A agonist, B-type natriuretic peptide (BNP), stimulates cGMP and shows anti-fibrotic, anti-hypertensive, and vasopressin-suppressive effects, potentially counteracting PKD pathogenesis. Here, we assessed the impacts of guanylyl cyclase A activation on PKD progression in a rat model of PKD. Sustained BNP production significantly reduced kidney weight, renal cystic indexes and fibrosis, in concert with suppressed hepatic cystogenesis in vivo . In vitro , BNP decreased cystic epithelial cell proliferation, suppressed fibrotic gene expression, and increased intracellular calcium. Together, our data demonstrate multifaceted effects of sustained activation of guanylyl cyclase A on polycystic kidney and liver disease. Thus, targeting the guanylyl cyclase A-cGMP axis may provide a novel therapeutic strategy for hepatorenal fibrocystic diseases.

Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV- 6-57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future.

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 (VL) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 VL. Previous work has demonstrated that the thermodynamic stability of native AL-09 VL is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 VL sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 VL forms an extensively ordered and β-strand-rich fibril structure. Furthermore, we demonstrate that the predominant β-sheet secondary structure and rigidity observed for in vitro prepared AL-09 VL fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils.

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. As such, the initial concentration of soluble amyloidogenic protein is expected to have a profound effect on the rate of fibril formation. In this work, we present in vitro evidence that fibril formation rates for AL light chains are affected by the protein concentration in a differential manner. De novo reactions of the proteins with the fastest amyloid kinetics (AL-09, AL-T05, and AL-103) do not present protein concentration dependence. Seeded reactions, however, exhibited weak protein concentration dependence. For AL-12, seeded and protein concentration dependence data suggest a synergistic effect for recruitment and elongation at low protein concentrations, while reactions of κI exhibited poor efficiency in nucleating and elongating preformed fibrils. Additionally, co-aggregation and cross seeding of κI variable domain (V

Urinary extracellular vesicles (uEVs) have been used as a biomarker tool for detection of low abundance immunoglobulin-based targets in uEVs with heterogeneous high protein mixtures. This protocol outlines a pre-incubation method to minimize non-specific secondary antibody binding. Additionally, we include our approach for standardization of uEV amounts used for sample comparison. We propose that these and knowledge of the detection limit for target proteins are strategies applicable to other laboratories working with complex heterogeneous samples.

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 (VL) relative to its germline counterpart, κI O18/O8 VL. 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. Currently, however, there is extremely limited structural information available which explicitly describes the residues that are involved in supporting the misfolded fibril structure. Here, we assign the site-specific 15N and 13C chemical shifts of the rigid core residues of AL-09 VL fibrils by solid-state NMR, reporting on the regions of the protein involved in the fibril as well as the extent of secondary structure.

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis.

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. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than de novo reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction.

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. In this report, we compare the stabilities and amyloidogenic properties of two light chains, an amyloid-associated protein AL-09 FL, and its germline protein κI O18/O8 FL (IGKV 1–33). We demonstrate that the thermal unfolding for both proteins is irreversible andand scan rate dependent, with similar stability parameters compared to their VL counterparts. In addition, the constant domain seems to modulate their amyloidogenic properties and affect the morphology of the amyloid fibrils. These results allow us to understand the role of the kappa constant domain in AL amyloidosis.

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. We start by giving a brief introduction on amyloid assembly and the advantages of using simple and readily available techniques to study aggregation. After an overview on preparation of protein to set up parallel experiments, we provide a systematic description of the in vitro techniques used to study aggregation in AL protein. Additionally, we thoroughly discuss the steps needed in our experience during the individual experiments for better reproducibility and data analysis.

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. We will discuss the role of cofactors and the mechanism of cellular damage. Last, we will review our recent findings on the high resolution structure of AL amyloid fibrils. AL amyloidosis is the best example of protein sequence diversity in misfolding diseases, as each patient has a unique combination of germline donor sequences and multiple amino acid mutations in the protein that forms the amyloid fibril.

Following the publication of this article, the authors noted that Patrick M. Vanderboom was inadvertently omitted from the author list. The correct author list is as follows: Sanjay Kumar, David Murray, Surendra Dasari, Paolo Milani, David Barnidge, Benjamin Madden, Patrick M. Vanderboom, Taxiarchis Kourelis, Bonnie Arendt, Giampaolo Merlini, Marina Ramirez-Alvarado, Angela Dispenzieri. Patrick M. Vanderboom is affiliated with the Mayo Clinic in Rochester, MN.

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. However, certain mutations either decrease (H32Y and H70D) or increase (R65S and Q96Y) the protein thermal stability. Interestingly, the most and the least stable mutants, Q96Y and H32Y, do not form amyloid fibrils under physiological conditions. Thus, Q96 and H32 are key residues for AL-12 stability and fibril formation and restoring them to the wild-type residues preclude amyloid formation. The mutants whose equilibrium is shifted to either the native or unfolded states barely sample transient partially folded states, and therefore do not form fibrils. These results agree with previous observations by our laboratory and others that amyloid formation occurs because of the sampling of partially folded states found within the unfolding transition (Blancas-Mejia and Ramirez-Alvarado, Ann Rev Biochem 2013;82:745-774). Here we provide a new insight on the AL amyloidosis mechanism by demonstrating that AL-12 does not follow the established thermodynamic hypothesis of amyloid formation. In this hypothesis, thermodynamically unstable proteins are more prone to amyloid formation. Here we show that within a thermal stability range, the most stable protein in this study is the most amyloidogenic protein.

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. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates.

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. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils. Methods We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils. Results We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease. Conclusions This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.

Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV- 6-57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future.

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as "direct activators" that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (KD = 26 ± 5 nM) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.

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. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.

Introduction: The clinical presentation and frequency of organ involvement are quite distinct in patients with IgM amyloidosis compared to patients with non-IgM amyloidosis, with less heart involvement and more nerve, soft tissue and lung involvement in IgM patients. It has been observed that immunoglobulin light chain variable region (IGVL) gene and gene family of the plasma cell clone impact phenotypic manifestations in AL amyloidosis, particularly organ tropism. (Kourelis et al. Blood. 2017;129). Given the variability in clinical features amongst patients with IgM vs. non IgM AL amyloidosis, we hypothesized that IGVL gene usage would be different between these two groups. Methods: Patients with newly diagnosed IgM and non-IgM amyloidosis seen at our institution from 01/2006 to 12/2015 were identified. IGVL gene usage was assessed by liquid chromatography tandem mass spectrometry (LC/MS/MS) as previously described. (Vrana et al Blood 2009;Dasari et al J of Proteome Res; Kourelis et al. Blood. 2017). Mass spectrometry data for IGVL analysis was obtained from clinically available data. LC/MS/MS was performed on available archival specimens when such data was not available clinically. Results: Patients with newly diagnosed amyloidosis who had sufficient mass spectrometry data on fat aspirate or tissue biopsy for IGVL gene usage identification were included (IgM AL, N=44 and Non-IgM AL, N=391). Clinical Features: As expected, differences were noted in baseline characteristics of patients with IgM vs. non-IgM AL amyloidosis. Patients with IgM amyloidosis were older (68 vs. 64 years, p=0.04) and more likely to have kappa as the involved light chain (34% vs. 25%, p=0.2), though the difference did not reach statistical significance. IgM amyloidosis patients had lower difference in involved and uninvolved free light chains (dFLC) (15.5 vs. 25.8 mg/dL, p=0.01) and lower NTProBNP levels (median: 1987 vs. 2655 pg/mL, p=0.02) compared with non-IgM amyloid patients. Presence of t(11;14) was less common by FISH (30% vs. 48%, p=0.1), though the difference did not reach statistical significance. Patterns of organ involvement were different, with heart involvement being less frequent in IgM patients (64% vs. 76%), while peripheral nerve (30% vs. 18%, p=0.05), soft tissue (41% vs. 21%, p=0.005) and lung involvement (7% vs. 3%, p=0.1) were more common in patients with IgM vs. non-IgM AL amyloidosis. There was no difference in liver (9% vs. 15%), kidney (50% vs. 51%) GI (23% vs. 25%) or autonomic nervous system involvement (18% vs. 14%). IGVL Gene Usage: As shown in Figure 1, IGVL usage differed across the two groups. In the lambda group, LV2-08 (14% vs. 2%, p Correlation Between IGVL Gene usage and Clinical Features: IGVL gene usage correlated with disease characteristics and differences in the clinical presentation of patients across the two groups. LV2-14 was more commonly seen in IgM amyloid patients (above). It has been previously associated with a higher frequency of peripheral nerve involvement (Kourelis et al. Blood 2017), which was more frequent in IgM group. LV2-14 is also associated with low dFLC levels, which were noted to be lower in the IgM AL cohort. LV1-44 has been associated with higher likelihood of cardiac involvement. LV1-44 was seen less frequently in the IgM amyloid patients, who also had lower rates of cardiac involvement. LV6-57 has been historically associated with higher rates of t(11;14). It was less common in IgM AL patients, who also had lower rates of t(11;14). Conclusions: IGVL gene usage differed significantly in patients with IgM vs. non-IgM amyloidosis, correlating with differences in disease characteristics and organ involvement. This is the first study to our knowledge to report IGVL gene usage differences in IgM vs. non-IgM AL amyloidosis. Differences in IGVL gene usage may explain the distinct clinical presentation seen in IgM amyloidosis, the biology of which has so far remained poorly understood. *SS and SD contributed equally Disclosures Dasari: The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Dispenzieri:Alnylam: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Pfizer: Research Funding; Janssen: Consultancy; Akcea: Consultancy; Intellia: Consultancy. Murray:The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding. Gertz:Prothena: Honoraria; Alnylam: Honoraria; Ionis: Honoraria; Janssen: Honoraria; Spectrum: Honoraria, Research Funding; Celgene: Honoraria.

The transthyretin (TTR) and immunoglobulin light chain proteins do not exist in isolation in cardiac amyloid (CA) plaques. Laser microdissection followed by mass spectrometry-based proteomic (LMD-MS) typing techniques can identify hundreds of co-deposited proteins in the "nano-environment" of amyloid deposits. Some, such as the "amyloid signature" proteins (SAP, APOE, APOA4, vitronectin, clusterin), are specific for amyloid plaques across different tissues and amyloid types. However, many of the remaining proteins are also abundant in the serum or normal tissues making it difficult to discern if they are unique to the amyloid plaque or part of the normal tissue background. We included 288 patients/samples with TTR CA (of which 53 were mutated) diagnosed by endomyocardial biopsy and 5 normal control samples obtained from 3 patients during autopsy. At the time of the meeting updated analyses including 168 cardiac light chain amyloid patients and 15 controls from patients with restrictive and hypertensive cardiomyopathy will be presented. Normalized spectral counts were used as a semi-quantitative measure of abundance. Proteins were considered part of the proteome if their abundance in the amyloid plaque was increased by 1.5-fold compared to normal controls (FDR p Of 2240 unique proteins identified across all samples, 42 were differentially expressed in amyloid plaques. These included a) signature proteins: SAP, APOE, APOA4, vitronectin, clusterin; b) serum proteins: albumin, b2-glycoprotein, 7 complement pathway proteins; c) matrix proteins: 2 collagen isoforms, fibulin 1, decorin, PCOLCE2, TIMP3; d) cytosketal and contractile proteins: 2 actin isoforms, MYL2, 8 myosin isoforms, 3 ankyrin isofroms, desmin; and "other" proteins: serpine2, MAGE-like protein-2, pleiotrophin, AMBP, SRPX and QSOX1). The signature and cytoskeletal proteins were the most abundant overall, however, proteins that were most increased compared to normal included (log2 fold change in parentheses): fibulin (24.5), PCOLCE2 (23.5), b2-glycoprotein (23.5), factor H related peptide-2 (24.2), serpine2 (24.7), mage-like protein-2 (22.8), pleiotrophin (23.8), QSOX1 (22.9), SRPX (23.5). B2 glycoprotein was more abundant in wild type TTR samples(p=0.003). APOE (p=0.002), fibulin (p=0.008) and serpine2 (p Our data shows that the TTR CA proteome includes signature, serum and cardiac tissue proteins with variable expression according to TTR mutation status and cardiac functional status, which could inform mechanisms of disease pathogenesis. Comparison with diseased controls may further clarify if this proteomic pattern is specific to CA. Figure Disclosures Dasari: The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Dispenzieri:Akcea: Consultancy; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding; Celgene: Research Funding; Alnylam: Research Funding.