Randal R. Ketchem

Solid-state nuclear magnetic resonance spectroscopy of uniformly aligned preparations of gramicidin A in lipid bilayers has been used to elucidate a high-resolution dimeric structure of the cation channel conformation solely on the basis of the amino acid sequence and 144 orientational constraints. This initial structure defines the helical pitch as single-stranded, fixes the number of residues per turn at six to seven, specifies the helix sense as right-handed, and identifies the hydrogen bonds. Refinement of this initial structure yields reasonable hydrogen-bonding distances with only minimal changes in the torsion angles.

We report here the cloning and characterization of four new members of the interleukin-1 (IL-1) family (FIL1δ, FIL1ε, FIL1ζ, and FIL1η, with FIL1 standing for “Family of IL-1”). The novel genes demonstrate significant sequence similarity to IL-1α, IL-1β, IL-1ra, and IL-18, and in addition maintain a conserved exon-intron arrangement that is shared with the previously known members of the family. Protein structure modeling also suggests that the FIL1 genes are related to IL-1β and IL-1ra. The novel genes form a cluster with the IL-1s on the long arm of human chromosome 2. We report here the cloning and characterization of four new members of the interleukin-1 (IL-1) family (FIL1δ, FIL1ε, FIL1ζ, and FIL1η, with FIL1 standing for “Family of IL-1”). The novel genes demonstrate significant sequence similarity to IL-1α, IL-1β, IL-1ra, and IL-18, and in addition maintain a conserved exon-intron arrangement that is shared with the previously known members of the family. Protein structure modeling also suggests that the FIL1 genes are related to IL-1β and IL-1ra. The novel genes form a cluster with the IL-1s on the long arm of human chromosome 2. interleukin accessory protein interleukin 1 receptor antagonist polymerase chain reaction probability density function lipopolysaccharide The cytokine interleukin-1 (IL-1)1 elicits a wide array of biological activities that initiate and promote the host response to injury or infection, including fever, sleep, loss of appetite, acute phase protein synthesis, chemokine production, adhesion molecule up-regulation, vasodilatation, the pro-coagulant state, increased hematopoiesis, and production and release of matrix metalloproteinases and growth factors (1.Dinarello C.A. Int. Rev. Immunol. 1998; 16: 457-499Crossref PubMed Scopus (682) Google Scholar). 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We have searched for novel members of the IL-1 family. We report here the sequences and some of the characteristics of four genes that appear to have descended from the same common ancestor as did IL-1α, IL-1β, IL-1ra, and IL-18. We propose that these novel molecules be designated FIL1δ, -ε, -ζ, and -η, with FIL1 being an acronym for Family of IL-1. The following details supplement the general descriptions given under “Results” for the cloning of the individual IL-1 family members. A 469-base pair single-stranded32P-labeled PCR the in was to a human in in of which to the by with PCR on from the same was to the A human in was a to the sequence in in in The from was to the of which the of the human of human to the of this was of this of the The structure of the gene was by of a PCR in which the predicted as from the and and from human the sequence for amino acid from amino acid The was in a from the A human to was sequences for structure modeling from the Protein J.R. O. T. M. J. PubMed Scopus Google Scholar). A sequence for the was based on that by (19.Bazan J.F. Timans J.C. Kastelein R.A. Nature. 1996; 379: 591Crossref PubMed Scopus (265) Google Scholar) for the IL-1s and IL-18, which by of and predicted the L. L. B. A. Protein Sci. 1998; 7: PubMed Scopus Google Scholar) that the for IL-1β and and and for the new IL-1 family members FIL1δ, FIL1ε, and the sequence of the new cytokines is to IL-1ra, a to the structure of the IL-1β appear by the so for A. L. F. H. M. 1995; PubMed Scopus Google Scholar) was to generate a family of for each a with in the the molecule probability density function by from to The structure with the PDF was a based on the PDF and that the was in the of and At this the for the to that A structure for each was by the a family by PDF with such as the members their The structure with the from the was as the the R.A. J.A. M.W. R. J.M. J. 1996; PubMed Scopus Google Scholar) and was that the are with The structure for FIL1δ, FIL1ε, and be by the was by cloning or of from the novel IL-1 so that the PCR another to that not The sequence of the PCR from was with the sequence in to the was the G. K. C. H. N. D. D. J.F. C. C. J. J. 1996; PubMed Scopus Google Scholar) from which of from the was PCR for the human of each novel IL-1 family by and by was in the following was was 1 was was a of 2 was the was to the for and to known on the for each gene are as FIL1δ, for of IL-1α, IL-1β, and from the and IL-1ra, and in I and II and the by PCR given in The to so that from and be some in a PCR The of an for each combination was by on with sequences for of novel IL-1 family member or some a PCR reaction was for and and are also in a new some a PCR reaction was for and and are also individual from human from and as M. M. G. J. 1994; PubMed Scopus Google Scholar, M. J.M. G. Blood. 1994; PubMed Google Scholar, D.L. W. T. C.A. R.J. Chin W. L. L. M. G. J. Immunol. PubMed Scopus Google Scholar). NK cells with 1 for 2 or T cells or with on or with the combination of and was in form as a of for or with 1 for 2 or cells or with a combination of for or cells with as for for 2 or of and of PCR and as IL-1 family as as IL-1β and IL-18 molecules, by of cells D. Cerretti D.P. Larsen A. L. March C. Dower S. Gillis S. Urdal D. Nature. PubMed Scopus Google Scholar). J.G. M. J. Shanebeck K. Grabstein K. S. A. Cosman D. D. EMBO J. 1994; PubMed Scopus Google Scholar) for and FIL1ζ, or a for and The for FIL1δ, and FIL1ζ, the sequence with was of the human chain signal IL-1β, with an to the of the mature form with was in IL-18 was as the mature form to the signal peptide in the C.J. E. T. M. S.D. S. Gayle R.B. Cerretti D.P. 1995; 10: Google Scholar). cells of and for with of a for It is of that FIL1δ, FIL1ε, and appear to be secreted from the cells the of signal peptide or and from the the and their The amino acid of the secreted was by The amino acid of the secreted is 2. does not appear to have of an signal peptide or prodomain in are a of cells, not appear in the so this is not a general to the of a of of also in the following of The of these is The novel IL-1 family in from cells, for binding to of the IL-1 receptor members 2 for general T. L. D. L. M. J. and M. L. J. in IL-1R type IL-1R AcP, IL-1R and as of was for 2 with of protein in and 1 a the was to a and with 1 of protein another of protein The was and the with of and and The was four with 1 of a and and in of 2 on and The four previously known members of the IL-1 family IL-1β, IL-1ra, and a amino acid common amino acid sequence the of which be as is any amino and that of the amino is that are in the family as on the sequence searched and sequences to novel IL-1 family as FIL1δ, FIL1ε, and A novel family as FIL1η, was originally in a of the sequence by the in the that was from an We searched for and an form of that contains the conserved family sequence in the A of the cloning and characteristics of each of the family members is given The and a with the previously known IL-1 are given in A of a that the known IL-1s but was not to The to the was and to the of an the known family this novel to a signal peptide a prodomain the A human was from a human as a The human sequence predicted an to that of from and the same predicted with for signal peptide or the from sequences not sequences appear to from in that be in sequence of the and have their gene is from least two A of a that a novel IL-1 family of the to the an that to the of an IL-1 but which not be in the The sequence was to a human and a was and the The sequence a pair with homology to the of was a the of this and a the of the acid human to the of this was and with sequence similarity to the and to IL-1 family members. the that the four to form a PCR and to the predicted from human for FIL1δ, the predicted contains a signal peptide a A third was in that to an that the a of the but of two in of a as as a that an that did with a and that for amino was named with family members suggests that a prodomain of some amino of that an the the and amino of the amino acid form 1 The sequences in the this and appear to be by for FIL1ζ, only of which to a was to the of the was by PCR that and the and to in a of The was in and We not the form of by the in any that by the form of was in which originally and but not in the The that form to be the for novel IL-1 family NK on the novel IL-1 family members. that the was for but not a that the was not for that as by PCR from a of by by the of an by PCR of an individual that of the gene was increased by the for was a of and the for human and are is a and are is an is an is a is a in that the PCR 2. The PCR for FIL1δ, FIL1ε, and especially from that for was in and in a new The on the novel IL-1 family members. that the was for but not a that the was not for that as by PCR from a of by by the of an by PCR of an individual that of the gene was increased by the for was a of and the for human and are is a and are is an is an is a is a in that the PCR 2. The PCR for FIL1δ, FIL1ε, and especially from that for was in and A of the sequence was originally in an P. J. R. Scholar). The sequence in this to the of an IL-1 which in the the of the IL-1 family members the sequence including the and the of the of a conserved in the IL-1 family searched for an that a more member of the family. PCR with from the of the sequence P. J. R. from and and only in and a from the and a from the of the sequence with the of the sequence and was and A was of the of of the sequence from family this the sequence for the of an IL-1 family as as a its PCR to a by of the of the sequence with this did a from human which the predicted acid The and the gene named Like and FIL1ε, does not an signal peptide or The novel members of the IL-1 family are as to another in sequence as are to the this of is in turn to that by the IL-1s any given pair of family members sequence that as being more to another are and also be in the in in which that IL-1α, IL-1β, and IL-18 form sequence FIL1ε, FIL1ζ, and form a and and form a The novel members be the sequence by (19.Bazan J.F. Timans J.C. Kastelein R.A. Nature. 1996; 379: 591Crossref PubMed Scopus (265) Google Scholar) sequence the IL-1 as by the IL-1α, IL-1β, IL-18, and IL-1ra, the mature peptide signal sequence or was for the FIL1ζ, the mature form was to with based on sequence and of predicted and this sequence was in a new The sequence the IL-1 as by the IL-1α, IL-1β, IL-18, and IL-1ra, the mature peptide signal sequence or was for the FIL1ζ, the mature form was to with based on sequence and of predicted and this sequence was The of FIL1δ, FIL1ε, and have as the of IL-1β and IL-1ra. The novel IL-1 member amino acid sequences with be which the IL-1β and the structure conserved for The of the FIL1δ, FIL1ε, and and and the in the the IL-1β and also from each The known genes of the IL-1 family a conserved of and their from a common The novel IL-1 family members here demonstrate the same The in the and the predicted to the structure of the IL-1α have this the of the family only the sequence IL-18, which to the and of the and to is more in IL-1α, and in FIL1ζ, the not of the of the family to not of the It is also more in the the FIL1ε, or of the The novel IL-1 family members have the cluster on human chromosome the and The this same At the of with the the novel and IL-1 genes appear to be We have the of the novel IL-1 family members in a of from as for that the novel family members are in the from cytokine to cytokine for each is also in a of II also from from and from PCR of individual about for the individual cytokines or for the family, is We to of each of the novel IL-1 family members in individual from from human and for a in was and by for the of FIL1δ, -ε, -ζ, and family members in and was some in these cells without was also in The only of the family members by T cells was of novel family members in not of IL-1 family members was by PCR of from of cells, as under not in a new of IL-1 family members was by PCR of from of cells, as under We any of the novel IL-1 family members to the known members of the IL-1 receptor family. from cells with each of the novel and with as as from cells with IL-18 as a with of the IL-1R family members type IL-1R AcP, and by of the protein G. The on and to any of the to to any of the IL-18 was to to this not We here the of novel genes that the of the IL-1 of the FIL1ζ, and genes as of IL-1α, IL-1β, IL-1ra, and IL-18 is based on sequence conserved amino acid only is of but the amino acid sequences modeling a predicted structure that is conserved with the known IL-1s is conserved in these new genes and is to that in the IL-1s as as IL-18 an of from a common with by gene the new IL-1 members are in the same of human chromosome that contains and IL-18 is the only member that does not to this The novel IL-1 family members are in a of and It is not to about to that to be the only of these cytokines in T cells, and of the family members are in and the of to these genes in as as the of PCR required to an in appear that are FIL1δ, FIL1ε, and be by in and cells, and to be from least two that regulation of in this family is It be that the new IL-1 members to members of the IL-1 receptor have to demonstrate this in of the known receptors and receptor It be that are as members of the IL-1R the with the IL-1s and IL-18, binding by two receptor subunits to be of is that these cytokines to a type of receptor. IL-18, for was to be of binding with to a protein that similarity to IL-1R family members D. Kim S.H. Fantuzzi G. Reznikov L.L. C.A. Rubinstein M. 10: Full Text Full Text PDF PubMed Scopus Google Scholar). The biological activity of the novel IL-1 family members to be IL-1α, IL-1β, and IL-18 are of activating gene that and promote It is to that FIL1δ, FIL1ε, FIL1ζ, and have the to the of the agonist IL-1s, and is that or more of the novel family members an antagonist this be that of binds to the type I IL-1R or to and not signaling by IL-1 or IL-18. It is also that the of these molecules to IL-1 about their receptor binding or the type of biological be by We Bonnert and Thomassen for on the in this for and and for for with and sequence and for with protein structure and for of and Arend for the

In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met(246), His(304), and His(427) in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxide-treated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.