158 research outputs found
Crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei reveals conformational plasticity at its C-terminus
BACKGROUND: Intracellular membrane fusion processes are mediated by the spatial and temporal control of SNARE complex assembly that results in the formation of a four-helical bundle, composed of one vesicle SNARE and three target membrane SNARE polypeptide chains. Syntaxins are essential t-SNAREs and are characterized by an N-terminal Habc domain, a flexible linker region, a coiled-coil or SNARE motif and a membrane anchor. The N-terminal Habc domain fulfills important regulatory functions while the coiled-coil motif, present in all SNAREs, is sufficient for SNARE complex formation, which is thought to drive membrane fusion. RESULTS: Here we report the crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei, s-syntaxin. Squid Habc crystallizes as a dimer and the monomer structure consists of a three-helical bundle. One molecule is strikingly similar to mammalian syntaxin 1A while the second one shows a structural deviation from the common fold in that the C-terminal part of helix C unwinds and adopts an extended conformation. CONCLUSION: Conservation of surface residues indicates that the cytosolic part of s-syntaxin can adopt an auto-inhibitory closed conformation that may bind squid neuronal Sec1, s-Sec1, in the same manner as observed in structure of the rat nSec1/syntaxin 1A complex. Furthermore, despite the overall structural similarity, the observed changes at the C-terminus of one molecule indicate structural plasticity in neuronal syntaxin. Implications of the structural conservation and the changes are discussed with respect to potential Habc domain binding partners such as Munc13, which facilitates the transition from the closed to the open conformation
Crystal structure of subunit VPS25 of the endosomal trafficking complex ESCRT-II
BACKGROUND: Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity. RESULTS: Here we report the crystal structure of Vps25 at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 [1,2]. CONCLUSIONS: Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex
Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction
We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the
variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the
chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region
of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated
oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments
into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig)
promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte
antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into
non-Ig-producing myeloma cells
VH-RELATED IDIOTOPES DETECTED BY SITE-DIRECTED MUTAGENESIS
The function of the CD4 cell surface protein as
coreceptor on T helper lymphocytes and as receptor
for HIV makes this glycoprotein a prime target for
an immune intervention with mAb. A detailed understanding
of the structural determinants on the
therapeutic CD4 mAb that are involved in Ag binding
or are recognized by anti-idiotypic mAb (anti-Id)
may be important for designing antibodies with optimal
therapeutic efficacy. Seven anti-Id raised
against the CD4 mAb M-T310 were selected from a
large panel with the intention to obtain CD4 mimicking
structures with specificity foHr IV gp120. The
selected anti-Id did not reacwt ith other CDCspecific
mAb cross-blocking M-T310. Among these, mAb MT404,
although having the same L chain as M-T310
and a VH region sequence differing onlya t 14 amino
acid positions, was not recognized by the anti-Id. MT310
H chain complexed with the J558L L chain
reacted with all anti-Id, thus demonstrating that the
recognized idiotopes are located within the VH region.
To identify the idiotopes of M-T310 seen by
the anti-Id, variants of M-T404 containing one or
more of the M-T3 1 O-derived substitutions were generated
by oligonucleotide-directed mutagenesis.
The reactivity pattern of the mutant proteins with
the anti-Id demonstrated that the idiotopes reside
within the complementarity determining region
(CDR) 2 and CDR3 loops of the VH region. A major
idiotope was definebdy a single amino acid in CDR2
that was recognized by three anti-Id, whereas the
four other anti-Id reacted with determinants of
CDR3. Although the performed amino acid substitutions
did influence the Id recognition, Ag binding
was not significantly affected, suggesting that none
of the anti-Id can be considered as a mimicry of the
CD4 A
Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction
We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the
variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the
chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region
of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated
oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments
into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig)
promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte
antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into
non-Ig-producing myeloma cells
EXPRESSION OF A FUNCTIONAL CHIMERIC lg-MHC CLASS II PROTEIN
composed of the a- and ß-chains of the MHC class I1
I-E molecule fused to antibody V regions derived
from anti-human CD4 mAb MT310. Expression vectors
were constructed containing the functional,
rearranged gene segments coding for the V region
domains of the antibody H and L chains in place of
the first domains of the complete structural genes
of the I-E a- and ß-chains, respectively. Celltsr ansfected
with both hybrid genes expressed a stable
protein product on the cell surface. The chimeric
molecule exhibited the idiotype of the antibody
MT310 as shown by binding to the anti-idiotypic
mAb 20-46. A protein of the anticipated molecular
mass was immunoprecipitated witha nti-mouse IgG
antiserum. Furthermore, human soluble CD4 did
bind to thetr ansfected cell line, demonstrating that
the chimeric protein possessed the binding capacity
of the original mAb. Thus, the hybrid molecule retained:
1) the properties of a MHC class I1 protein
with regardt o correct chain assembly and transport
to the cell surface: as well as 2) the Ag binding
capacity of the antibody genes used. Thgee neration
of hybrid MHC class I1 molecules with highly specific,
non-MHC-restricted bindingc apacities will be
useful for studying MHC class 11-mediated effector
functions such as selection of the T cell repertoire
in thymus of transgenic mice
Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the a-chain of the human interleukin-2 receptor
The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of
them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized
two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs
are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human
complement, the individual Abs are negative; however, when used in combination, complement activation was performed.
When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display
this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by
combination of an anti-CD4 mAb with one directed against the a-chain of the human IL2 receptor, nearly 100% inhibition
of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4
mAb and an anti-IL2Rcc chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating
that this mAb cocktail could be a new strategy for immunosuppressive therapy
Роль метода электрофоретического осаждения в создании биокомпозита на основе слоев гидроксиапатити и наночастиц серебра
Работа посвящена созданию многофункционального биокомпозита, состоящего из покрытия на основе гидроксиапатита (ГА) и наночастиц серебра с использованием высокотехнологичных методов обработки поверхности. Высокочастотное магнетронное распыление использовалось для получения слоев ГА покрытия с различной толщиной и структурой на титане и наночастицах серебра. Для получения антибактериального слоя наночастиц серебра использовался метод электрофоретического осаждения. Наночастицы серебра имели сферическую форму с диаметром 70±20 нм и[zeta] -потенциалом -20 мВ. Дифракционные картины биокомпозитов выявили пики кристаллического ГА и серебра (Ag). Так же установлено, что наночастицы серебра являются кристаллическими с размером кристаллитов 14 нм
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Phosphorylated T Cell Receptor ζ-Chain and ZAP70 Tandem SH2 Domains Form a 1:3 Complex In Vitro
The ζ polypeptide is part of the T cell antigen receptor (TCR). The ζ-chain contributes to efficient cell-surface expression of the TCR and accounts for part of its signal transduction capability. TCR recognition triggers a complex set of events that result in cellular activation. The protein tyrosine kinase (PTK) Lck phosphorylates the ζ-chain, which in turn associates with another PTK, ZAP70, and stimulates its phosphorylation activity. Here we report the expression of the intracellular part of the ζ-chain and its biochemical characterization. The recombinant protein does not dimerize by itself in solution. Circular-dichroic analysis reveals a random coil conformation. ζ, phosphorylated using recombinant Lck, associates with recombinant ZAP70 tandem-SH2 domains. All three T cell activation motifs in ζ bind ZAP70 tandem-SH2 domains in vitro, forming a 1:3 complex. This result extends the picture, derived from earlier studies, of a mechanism for signal amplification.Molecular and Cellular Biolog
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