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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. Our 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. Here we discuss how inclusive speaker selection fosters better scientific environments for all and 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.

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.

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 < 0.001). Heart (56% vs. 73%, p = 0.002) and >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 MYD88 L265P and CXCR4 WHIM 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 < 0.001; MYD88 L265P mutation: 84% (27/32) vs. 0% (0/14), p < 0.001; CXCR4 mutation: 29% (8/28) vs. 0% (0/14), p = 0.04. Overall survival was shorter in IgM AL when stratified by Mayo 2012 stage; stage 1/2 (59 vs. 125.9 months, p = 0.003) and stage 3/4 (6.5 vs. 12.9 months, p = 0.075), likely due to lower hematologic response rates (6 months: 39% vs. 59%, p = 0.008). We characterized two subtypes of IgM amyloidosis (LPL/PPCN). This can aid in therapeutic decision-making, with treatment directed at the clonal disease.

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.

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.

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., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., (© 2017 Wiley Periodicals, Inc.)

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 L ) relative to its germline counterpart, κI O18/O8 V L . 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 15 N and 13 C chemical shifts of the rigid residues of AL-09 V L 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)-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 L ) 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 L . Previous work has demonstrated that the thermodynamic stability of native AL-09 V L 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 V L sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 V L 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 V L 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 L ) and the κI full length (FL) light chain indicate that the presence of the constant domain in κI FL modulates fibril formation, facilitating the recruitment of κI V L . Together, these results indicate that the dominant process in fibril formation varies among the AL proteins tested with a differential dependence of the protein concentration.

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. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect., Methods and Results: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 μg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1., Conclusions: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

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 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., (© 2015 The Protein Society.)

Amyloid formation occurs when a precursor protein misfolds and aggregates, forming a fibril nucleus that serves as a template for fibril growth. Glycosaminoglycans are highly charged polymers known to associate with tissue amyloid deposits that have been shown to accelerate amyloidogenesis in vitro. We studied two immunoglobulin light chain variable domains from light chain amyloidosis patients with 90% sequence identity, analyzing their fibril formation kinetics and binding properties with different glycosaminoglycan molecules. We find that the less amyloidogenic of the proteins shows a weak dependence on glycosaminoglycan size and charge, while the more amyloidogenic protein responds only minimally to changes in the glycosaminoglycan. These glycosaminoglycan effects on fibril formation do not depend on a stable interaction between the two species but still show characteristic traits of an interaction-dependent mechanism. We propose that transient, predominantly electrostatic interactions between glycosaminoglycans and the precursor proteins mediate the acceleration of fibril formation in vitro., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pneumocystis organisms are opportunistic fungal pathogens that cause significant pneumonia in immune-compromised hosts. Recent evidence has suggested that Pneumocystis carinii exists as separate mating types, and expresses and regulates proteins that govern meiosis and progression of the life cycle. This study was undertaken to investigate the activity of three life cycle-regulatory proteins in Pneumocystis, including two proteins essential in mating signaling, and a putative meiotic regulator, to determine the conditions under which they are most active. This study used V5/HIS-tagged PCRan1p, PCSte20p, and PCCbk1, purified from Saccharomyces cerevisiae strain, INVSC, as well as an in vitro Escherichia coli protein expression system to determine the optimal expression conditions of each protein in the presence of varying pH, temperature, and metal ions. These studies demonstrate an atypical enzymatic activity in PCRan1p, whereby the kinase was most active in the environmental conditions between 10 and 25 degrees C, compared with a dramatic reduction in activity above 30 degrees C, temperatures typically found within mammalian hosts. Circular dichroism and fluorescence spectroscopy suggest that PCRan1p becomes partially unfolded at 25 degrees C, leading to its most active conformation, whereas continued unfolding as temperature increases results in strongly suppressed activity. These studies suggest that, in vivo, while under conditions within the mammalian lung (typically 37 degrees C), PCRan1p kinase activity is largely suppressed, allowing better conditions for the activation of meiosis, whereas in ex vivo environments, PCRan1p kinase activity increases to arrest progression of the life cycle until conditions become more favorable.

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel beta-helix with three adjoining segments of anti-parallel beta-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete beta-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel beta-sheet interactions between neighboring beta-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, beta-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders accompanied by a monoclonal gammopathy. Monoclonal free light chains (FLCs) are precursors of the pathological light chain tissue deposits that are fibrillar in AL and granular in NALCDD. However, direct biochemical examination of plasma FLC precursors, which would allow comparison and better understanding of these two diseases, is still lacking. In this study, we examined FLCs in plasma of patients with AL and NALCDD by employing separation on Sep-PaK C18 cartridges, micro-preparative electrophoresis, Western blotting and mass spectrometry. Comparative analysis of AL versus NALCDD and control plasma samples showed new evidence of increased level and heterogeneity of circulating disulfide-bound FLC species in AL. In addition to full length monomers comprising the disulfide-linked FLCs, the monoclonal disulfide-bound FLC fragments were typically revealed in AL plasma. We hypothesized that enhanced disulfide binding of FLCs in AL interferes with their normal clearance and metabolism, which in turn might play a role in amyloid formation. The applied methods might be useful to diagnose or predict the pathological form of the disease and shed light on the mechanisms involved in light chain aggregation in tissues.

Purpose: The lack of an in vivo diagnostic test for AD has prompted the targeting of amyloid plaques with diagnostic imaging probes. We describe the development of a contrast agent (CA) for magnetic resonance microimaging that utilizes the F(ab')2 fragment of a monoclonal antibody raised against fibrillar human Abeta42, Methods: This fragment is polyamine modified to enhance its BBB permeability and its ability to bind to amyloid plaques. It is also conjugated with a chelator and gadolinium for subsequent imaging of individual amyloid plaques, Results: Pharmacokinetic studies demonstrated this 125I-CA has higher BBB permeability and lower accumulation in the liver and kidney than F(ab')2 in WT mice. The CA retains its ability to bind Abeta40/42 monomers/fibrils and also binds to amyloid plaques in sections of AD mouse brain. Intravenous injection of 125I-CA into the AD mouse demonstrates targeting of amyloid plaques throughout the cortex/hippocampus as detected by emulsion autoradiography. Incubation of AD mouse brain slices in vitro with this CA resulted in selective enhancement on T1-weighted spin-echo images, which co-register with individual plaques observed on spatially matched T2-weighted spin-echo image, Conclusions: Development of such a molecular probe is expected to open new avenues for the diagnosis of AD.

Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kappaI O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kappaI O18/O8 dimer interface. The three non-conservative mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kappaI O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO(4)(2-) conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.

Background: The presence of monoclonal immunoglobulin free light chains (FLC) in the serum is commonly associated with the gammopathies, including multiple myeloma, systemic light chain amyloidosis and non-amyloid light chain deposition disease. Although a sensitive nephelometry-based assay is used for quantification of serum FLC and patient follow-up, this method does not provide information regarding the biochemical properties of these proteins. The present study focused on the development of the procedure for isolation and biochemical characterization of monoclonal FLC in small plasma specimens from patients with these disorders., Methods: Methods used in this study were ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein elution from gel and support membranes, dialysis, lyophilization, isoelectric focusing (IEF) and Western blotting., Results: The isolation, concentration and partial purification of FLC was based on micro-preparative SDS-PAGE employing analytical scale gels. For the determination of the isoelectric point of FLC, the developed protocol included consecutive IEF, electrotransfer of IEF-separated proteins onto and elution from support membranes, and their analysis by SDS-PAGE-based Western blotting. The procedures, which require only 20-50 microL of starting plasma, allow biochemical characterization of the monomeric, dimeric and truncated forms of FLC, including their charge properties., Conclusions: The developed procedure may be applied to reveal distinguishing chemical features of FLC in serum, which could be important in predicting the pathologic form of disease, and in yielding information to better understand the mechanism(s) involved in the deposition of light chains in tissues.

The tumor suppressor protein p53 is mutated in over half of human cancers. Despite 25 years of study, the complex regulation of this protein remains unclear. After serendipitously detecting RNA binding by p53 in the yeast three-hybrid system (Y3H), we are exploring the specificity and function of this interaction. Electrophoretic mobility shift assays show that full-length p53 binds equally to RNAs that are strongly distinguished in the Y3H. RNA binding blocks sequence-specific DNA binding by p53. The C-terminus of p53 is necessary and sufficient for strong RNA interaction in vitro. Mouse and human C-terminal p53 peptides have different affinities for RNA, and an acetylated human p53 C-terminal peptide does not bind RNA. Circular dichroism spectroscopy of p53 peptides shows that RNA binding does not induce a structural change in the p53 C-terminal peptide, and C-terminal peptides do not detectably affect the structure of RNA. These results demonstrate that p53 binds RNA with little sequence specificity, RNA binding has the potential to regulate DNA binding, and RNA-p53 interactions can be regulated by acetylation of the p53 C-terminus.

Light-chain amyloidosis (AL) is characterized by immunoglobulin light-chain fragments aggregating into amyloid fibrils that deposit extracellularly in vital organs such as the kidney, the heart, and the liver, resulting in tissue degeneration and organ failure, leading to death. Cardiac involvement is found in 50% of AL patients and presents the most severe cases with a life expectancy of less than a year after diagnosis. In this study, we have characterized the variable domain of a cardiac AL patient light chain called AL-09. AL-09 folds as a beta-sheet and is capable of forming amyloid fibrils both in the presence of sodium sulfate and in self-seeded reactions under physiological conditions. Glycosaminoglycans such as dermatan sulfate and heparin promote amyloid formation of self-seeded AL-09 reactions, while the glycosaminoglycan chondroitin sulfate A stabilized oligomeric intermediates and did not elongate the preformed fibrils (nucleus) present in the reaction. Finally, the histological dye Congo red, known to bind to the cross beta-sheet structure of amyloid fibrils, inhibits AL-09 amyloid fibril formation in the presence of sodium sulfate and in self-seeded reactions. This paper provides insight into the impact of different reagents on light-chain stability, structure, amyloid fibril formation, and inhibition.

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.