Role Of Amino Acid Sequence Research Articles

Sequence-dependent model of allosteric communication.

The omnipresence and diversity of allosteric regulation in proteins and protein associations complemented by the potential for the design of allosterically acting biologics and drugs call for the development of a new generation of computational models for the analysis of allostery and rational engineering/design of desired signaling and effector molecules determining it. One of the most important challenges is the consideration of the role of amino acid sequence in forming the protein's allosteric communication, including the mode and strength of the allosteric signal that is communicated to the regulated functional site. Here, we present the network-based model with a sequence dependence added in consideration of allosteric communication by combining the structure-based statistical mechanical model of allostery with the Miyazawa-Jernigan residue-residue potential. Applying the model in the analysis of five classical allosteric proteins, we found that it is necessary to consider the following two major determinants: (i) the free energy exerted by the allosteric site on the regulated one and (ii) the background (average) change in dynamics of the overall structure. We show that working together these two components determine the allosteric modulation, calling one to study their dependence on structures, oligomerization states, and sequence divergence in different proteins.

Abstract 3085: Role of amino acid sequences within the N terminal domain in regulating AF1-mediated glucocorticoid receptor activity

Abstract The steroid hormone receptors (SHRs) play an important role in many physiological and pathological processes including endocrine cancers. However, the precise mechanisms by which these SHRs pass signals from a hormone to control gene expression remain a central unresolved problem, which has limited SHRs therapeutic utilities in a cell/tissue specific manner. Role of the activation function-1 (AF1) in this process is of immense importance as it provides platforms for interactions of specific co-regulatory proteins, and thereby dictates the final outcome SHR-regulated target gene expression. The AF1 located in the N-terminal domain (NTD) plays a critical role in regulating cell/tissue-specific effects of SHRs. However, the exact mechanism of this action is not known. It has been suggested that cell/tissue-specific activities of SHRs may be due to AF1 conformations and subsequent coactivator interactions. The SHRs’ NTD/AF1 has been reported to exist in an intrinsically disorder conformation and undergoes disorder-order transition through intra- and inter- molecular allosteric regulations. In the study, we tested whether the flanking sequences around AF1 affect glucocorticoid receptor (GR) activity due, in part, to conformational changes in otherwise ID AF1 that can facilitate its interaction with critical coregulatory proteins such as TATA Box-Binding Protein (TBP) and steroid receptor coactivator-1 (SRC-1). Using biophysical techniques, we found that placing AF1 immediately upstream from the DNA binding domain (DBD) results into acquisition of a relatively ordered conformation in GR AF1. Further analyses revealed that removing amino acid sequences (1-76) prior to AF1 or removal of amino acids (263-420) between AF1 and DBD differentially regulates AF1-mediated GR activity. The results from this study may provide a potential mechanism for cell-dependent differing GR activities as well as other SHRs and thereby, our results may provide additional SHR selectivity needed to target cell-tissue specific gene regulations in current endocrine-based therapies. Citation Format: Shagufta H. Khan, Raj Kumar. Role of amino acid sequences within the N terminal domain in regulating AF1-mediated glucocorticoid receptor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3085.

Sars-Cov-2 Spike protein function prediction using a convolutional neural network ensemble

Introduction: SARS-CoV-2 has become a worldwide pandemic that affects all aspects of life; therefore, numerous organizations and open exploration foundations focus their efforts on research for viable therapeutics. Given past experiences and involvement in SARS, the essential focus has been the Spike protein, considered as the perfect objective for COVID-19 immunotherapies. Most of the vaccines being developed target the spike proteins because this protein covers the virus and helps it invade human cells. Methods: Applications of deep neural network is a quickly expanding field now reaching many areas including proteomics. Results: To be precise, convolutional neural networks have been used for identifying the functional role of amino acid sequences, because of its ability to give nearly accurate results for multi-label classification problems. Here we present a modified convolutional deep learning model that can identify if a given amino acid sequence is a spike protein or not based on the length of the sequence and the function of the protein, that will be done with a short execution time and a relatively small error rate.
 Conclusion: CNN is an efficient tool at supervised multilabel classification problems

Discovery of Surfactant-Like Peptides from a Phage-Displayed Peptide Library.

Peptides with specific affinities for various materials have been identified in the past three decades and utilized in materials science and engineering. A peptide’s capability to specifically interact with materials is not naturally derived but screened from a biologically constructed peptide library displayed on phages or cells. To date, due to limitations in the screening procedure, the function of screened peptides has been primarily limited to the affinity for target materials. Herein, we demonstrated the screening of surfactant-like peptides from a phage-displayed peptide library. A screened phage clone displaying a peptide showed high activity for accumulating at emulsion surfaces with certain assembled structures, resulting in stable emulsions. The surface tension for the solution of the chemically synthesized peptide decreased with increasing peptide concentration, demonstrating certain surface activity, which corresponded to the ability to decrease the surface tension of liquids (e.g., water), owing to the accumulation of molecules at the air–liquid or liquid–liquid interface. Peptides with a randomized sequence did not lower the surface tension, indicating the essential role of amino acid sequences in surface activity. Our strategy for identifying novel functional peptides from a phage-displayed peptide library can be used to expand the applicability of peptidyl materials and biosurfactants.

【原著】 ナノディスクのサイズ制御と安定性における アポA–Iへリックス構造の役割

Nanodiscs consist of phospholipids and scaffolding amphipathicα–helical proteins such as apolipoprotein A–I (apoA–I). To better understand the scaffolding function of apoA–I in nanodiscs, we examined the role of amino acid sequences in apoA–I to control the size and stability of nanodiscs using a series of deletion variants lacking different regions along the molecule. In cholate dialysis method, all apoA–I variants formed 1–palmitoyl–2–oleoyl phosphatidylcholine nanodiscs with diameters of 9～11 nm, in which the C–terminal deleted variant exhibited a poor ability to form nanodiscs. The nanodiscs of the C–terminal deleted variant were shown to be kinetically less stable compared to WT apoA–I nanodiscs, consistent with the importance of the C–terminal hydrophobic region in the formation of stable nanodiscs by apoA–I. Importantly, the correlation of the protein–to–phospholipid composition ratio with the number ofα–helical residues in the apoA–I variants on nanodiscs indicates that the lipid-holding capacity of apoA–I largely depends on the number ofα–helical residues on nanodiscs. These results imply the importance of theα– helix–forming ability of apoA–I in not only the energetics of the formation of nanodiscs but also the lipid–holding capacity of nanodiscs.

Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions

The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.

Aggregation of peptides in the tube model with correlated sidechain orientations

The ability of proteins and peptides to aggregate and form toxic amyloid fibrils is associated with a range of diseases including BSE (or mad cow), Alzheimer's and Parkinson's Diseases. In this study, we investigate the the role of amino acid sequence in the aggregation propensity by using a modified tube model with a new procedure for hydrophobic interaction. In this model, the amino acid sidechains are not considered explicitly, but their orientations are taken into account in the formation of hydrophobic contact. Extensive Monte Carlo simulations for systems of short peptides are carried out with the use of parallel tempering technique. Our results show that the propensity to form and the structures of the aggregates strongly depend on the amino acid sequence and the number of peptides. Some sequences may not aggregate at all at a presumable physiological temperature while other can easily form fibril-like, β-sheet struture. Our study provides an insight into the principles of how the formation of amyloid can be governed by amino acid sequence.

Role of Sequence and Structural Polymorphism on the Mechanical Properties of Amyloid Fibrils

Amyloid fibrils playing a critical role in disease expression, have recently been found to exhibit the excellent mechanical properties such as elastic modulus in the order of 10 GPa, which is comparable to that of other mechanical proteins such as microtubule, actin filament, and spider silk. These remarkable mechanical properties of amyloid fibrils are correlated with their functional role in disease expression. This suggests the importance in understanding how these excellent mechanical properties are originated through self-assembly process that may depend on the amino acid sequence. However, the sequence-structure-property relationship of amyloid fibrils has not been fully understood yet. In this work, we characterize the mechanical properties of human islet amyloid polypeptide (hIAPP) fibrils with respect to their molecular structures as well as their amino acid sequence by using all-atom explicit water molecular dynamics (MD) simulation. The simulation result suggests that the remarkable bending rigidity of amyloid fibrils can be achieved through a specific self-aggregation pattern such as antiparallel stacking of β strands (peptide chain). Moreover, we have shown that a single point mutation of hIAPP chain constituting a hIAPP fibril significantly affects the thermodynamic stability of hIAPP fibril formed by parallel stacking of peptide chain, and that a single point mutation results in a significant change in the bending rigidity of hIAPP fibrils formed by antiparallel stacking of β strands. This clearly elucidates the role of amino acid sequence on not only the equilibrium conformations of amyloid fibrils but also their mechanical properties. Our study sheds light on sequence-structure-property relationships of amyloid fibrils, which suggests that the mechanical properties of amyloid fibrils are encoded in their sequence-dependent molecular architecture.

Experimental and Simulation Studies on the Mechanical Properties of Sumo Proteins

Protein structure plays an important role in determining its mechanical stability. In order to understand the role of amino acid sequence on the mechanical stability, mechanical unfolding experiments were performed using atomic force microscope based single-molecule force spectroscopy (SMFS) on structurally homologous proteins - ubiquitin, SUMO1, and SUMO2. SUMOs are found to be mechanically weaker than ubiquitin, in spite of their high structural homology, when pulled along N-C direction [1]. The unfolding forces of these proteins follow the same trend as the number of sidechain contacts within them. The energy landscape parameters reveal that SUMOs are more flexible than ubiquitin. We have performed steered molecular dynamics simulations on these proteins to gain atomistic insights into their unfolding pathways. The hydrogen bonds in the mechanical clamp between the terminal β-strands of these proteins are ruptured at the transtion state and proteins unfolded in an all-or-none fashion. The interactions between the β2 strand and the α-helix also weaken at the transition state. We examine the different types of inter-resiude interactions in these proteins to rationalize the lower mechanical stability of SUMOs relative to ubiquitin. Role of water penetration during mechanical unfolding was also investigated.We have further studied the effect of ligand binding on the mechanical stability of SUMO proteins using SMFS in presence of short peptides derived from the binding motifs of SUMO targets. The unfolding force of SUMO1 increased from ∼130pN to ∼170pN upon ligand binding. Dependence of the unfolding force on the pulling speed was higher for SUMO1 bound to the peptides than the unbound protein. The flexibility of SUMOs decreased upon ligand binding suggesting a possible role of SUMO flexibility in SUMOylation.[1] Kotamarthi HC, Sharma R, Ainavarapu SRK, Biophys. J (2013), 104, p2273.

Folding of proteins in presculpted free energy landscape

Recent studies of the tube model of protein have indicated that the free energy landscape of proteins is presculpted by symmetry of the protein backbone and geometrical constraints played by the hydrogen bonds. In this study, we investigate the role of amino acid sequences in the folding of proteins. We consider two models that are differed by sequence specificity: the tube HP model with hydrophobic (H) and polar (P) sequences, and the tube Go model with native-centric contact potentials. Monte Carlo simulations are carried out for two sequences of length of 48 amino acids, whose ground states are a three-helix bundle and a GB1-like structure. The results show that folding inthe Go model is more cooperative than in the HP model. In the HP model the collapse transition and the folding transition are separated, whereas in the Go model the two transitions coincide.

The Role of Amino Acid Sequence in the Self-Association of Therapeutic Monoclonal Antibodies: Insights from Coarse-Grained Modeling

Coarse-grained computational models of therapeutic monoclonal antibodies and their mutants can be used to understand the effect of domain-level charge-charge electrostatics on the self-association phenomena at high protein concentrations. The coarse-grained models are constructed for two antibodies at different coarse-grained resolutions by using six different concentrations. It is observed that a particular monoclonal antibody (hereafter referred to as MAb1) forms three-dimensional heterogeneous structures with dense regions or clusters compared to a different monoclonal antibody (hereafter referred to as MAb2) that forms homogeneous structures without clusters. The potential of mean force (PMF) and radial distribution functions (RDF) plots for the mutants (hereafter referred to as M1, M5, M7, and M10) show trends consistent with previously reported experimental observation of viscosities. The mutant referred to as M6 shows strongly attractive interactions that are consistent with previously reported negative second virial coefficients (B(22)) obtained from light-scattering experiments (Yadav et al. Pharm. Res. 2011, 28, 1750-1764; Yadav et al. Mol. Pharmaceutics. 2012, 9, 791-802). Clustering data on MAb1 reveal a small number of large clusters that are hypothesized to be the reason for the high experimental viscosity. This is in contrast with M6 (that differs from MAb1 in only a few amino acids), where cluster analysis reveals the formation of a large number of smaller clusters that is hypothesized to be the reason for the observed lower viscosity. The coarse-grained representations are effective in picking up differences based on local charge distributions of domains to make predictions on the self-association characteristics of these protein solutions.

Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation, Cell Fusion, and Pathogenicity

To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates. Methods We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6. Results Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate. Conclusions The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion. General significance Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.

Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding.

Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.

Phosphorylation and putative ER retention signals are required for protein kinase A-mediated potentiation of cardiac sodium current.

Activation of protein kinase A (PKA) increases Na+ current derived from the human cardiac Na+ channel, hH1, in a slow, nonsaturable manner. This effect is prevented by compounds that disrupt plasma membrane recycling, implying enhanced trafficking of channels to the cell membrane as the mechanism responsible for Na+ current potentiation. To investigate the molecular basis of this effect, preferred consensus sites (serines 483, 571, and 593) and alternative sites phosphorylated by PKA in the rat heart isoform (serines 525 and 528) were removed in the I-II interdomain linker, a region in the channel previously implicated in the PKA response. Our results demonstrate that the presence of either serine 525 or 528 is required for Na+ current potentiation. The role of amino acid sequences that can mediate channel-protein interactions was also examined. Removal of a PDZ domain-binding motif at the carboxy terminus of hH1 did not alter the PKA response. The I-II interdomain linker of the channel contains 3 sites (479RKR481, 533RRR535, and 659RQR661) with the sequence RXR, a motif known to mediate retention of proteins in the endoplasmic reticulum (ER). The PKA-mediated increase in Na+ current was abolished when all 3 sites were eliminated, with RRR at position 533 to 535 primarily responsible for this effect. These results demonstrate that both alpha-subunit phosphorylation and the presence of putative ER retention signals are required for the PKA-mediated increase in cardiac Na+ current, an effect that likely involves interaction of the I-II interdomain linker with other proteins or regions of the channel.

Evaluation of molecular visualization software for teaching protein structure differing outcomes from lecture and lab: Differing outcomes from lecture and lab

AbstractIn this study we measured the learning outcomes resulting from using molecular visualization software in lecture and in the teaching laboratory of a large introductory‐level undergraduate biology majors' course. The study was initially carried out in the Fall semester of 1999; the results of this study were used to devise an expanded laboratory component that was evaluated in a second study carried out in the Fall of 2000. In both studies, students (n = 175 and 161) attended two 50‐min lectures that used molecular visualization software to explain protein structure and function and the gene‐protein connection. Students also used this software during one 3‐h laboratory session as a tool for exploring these topics. Students completed open‐ended pre‐ and post‐surveys that involved a related but unfamiliar task. Survey responses were scored for correctness, as well as by the type(s) of explanations used in the response. We found the following eight types of responses that students employed to explain protein structure and function: genetics, protein structure, chemical interactions, amino acid sequence, purpose/teleology, extrinsic factors, miscellaneous, and none. In both studies, the frequencies of correct answers, as well as the frequencies of each response type, showed significant changes as a result of lecture and/or lab. The effects of lecture were highly similar in both studies. The changes in the expanded lab resulted in significant changes in outcome. Overall, the curriculum effectively communicated several core concepts in protein biochemistry and expanded the conceptual “toolkit” that students applied to problems of protein structure and function. Lecture increased students' understanding of the role of amino acid sequence, whereas lab tended to increase their understanding of three‐dimensional structure and the gene‐protein connection. Our results demonstrate that exposure to molecular visualization, even for a relatively brief time, can improve students' understanding of protein structure and function. In addition, we demonstrate the differing and largely non‐overlapping effects of lecture and lab, suggesting that effective use of molecular visualization should involve both types of activities.

A Designed System for Assessing How Sequence Affects α to β Conformational Transitions in Proteins

The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study alpha to beta conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an alpha-helical leucine zipper and a beta-hairpin. The parent peptide, Template-alpha, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce beta-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-alphaT. Unlike the parent peptide, Template-alphaT underwent a heat-inducible switch to beta-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions to amyloid, destabilization of the leucine-zipper form of Template-alphaT did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative beta-hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Template-alpha, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favor beta-structure promote amyloid formation.

Interaction of Hypericin with Serum Albumins: Surface-enhanced Raman Spectroscopy, Resonance Raman Spectroscopy and Molecular Modeling Study¶

Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1-H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp. Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 A between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences.

Multiple factors determine the selection of the ectodomain cleavage site of human cell surface macrophage colony-stimulating factor.

Human cell surface macrophage colony-stimulating factor (CSF-1256, M-CSF alpha) is converted to a soluble growth factor by a regulated proteolytic cleavage process at amino acid residues 157-159. We have previously shown that multiple factors specified by the juxtamembrane region determine the cleavage efficiency [Deng, P., Rettenmier, C. W., and Pattengale, P. K. (1996) J. Biol. Chem. 271, 16338-16343]. In the present paper, we studied the effect of various deletion, insertion, and substitution mutations at or near the cleavage site on both the number and size of cleaved CSF-1(256) products to identify the mechanisms by which the cleavage sites are selected. Deletion of regions 161-162 or 163-165, C-terminal to the cleavage site, as well as deletion of region 150-156, N-terminal to the cleavage site, each yielded a single cleavage product that was smaller than that derived from the wild type (WT). In these experiments cleavage apparently occurred at a specific distance from the transmembrane domain. Insertion of three additional residues between the normal cleavage site and the transmembrane domain yielded one major product that was the same size as the processed form of WT CSF-1(256). In this case the selection of the cleavage site was apparently determined by the amino acid sequence of the juxtamembrane region rather than by the distance from the transmembrane domain. Other amino acid substitutions at the cleavage site caused changes in cleavage site selection, providing additional evidence for the role of amino acid sequence in cleavage site selection. Finally, a comparison of cleavage site selection in the presence and absence of tunicamycin treatment showed that N-glycosylation of certain mutant forms of CSF-1(256) sterically interfered with protease accessibility, which in turn had an effect on the selection of the site used for cleavage. Taken together, these results indicate that cleavage site selection is determined by the amino acid sequence of the juxtamembrane region, the distance of the site from the transmembrane domain, and steric accessibility of the protease.

Domain Stability in Immunoglobulin Light Chain Deposition Disorders

Publisher Summary This chapter begins with a brief introduction to the general problem of protein deposition, with a review of possible molecular mechanisms. Subsequent sections describe results on the analysis of light chain aggregation and deposition in several forms of human disease and in Escherichia coli (E. coli) expression. The chapter reviews some general factors that play roles in the mechanism of amyloid deposition in vivo. Clearly, both physiological and molecular factors are important. The challenge has been and continues to be in defining how the relevant factors together control the timing, tissue location, and medical consequences of amyloid deposition. A major role for mutational effects on domain stability as a determinant of susceptibility to in vivo deposition of light chain V L domains is discussed. The potential importance of domain stability warrants a consideration here of the role of amino acid sequence in V L and light chain folding stability. Site directed mutagenesis and recombinant expression techniques can now be used, however, to make point mutants in the light chain or the V L domain to isolate and test suspected amyloidogenic mutations.