2,413 research outputs found
Activation of human natural killer cells by Plasmodium falciparum
The
purpose
of
work
described
in
this
thesis
was
to
(i)
determine
the
contribution
of
innate
immune
responses
to
the
early
pro-inflammatory
cytokine
response
to
Plasmodium
falciparum,
(ii)
describe
the
kinetics
and
cellular
sources
ofIFN-y
production
by
human
PBMC
in
response
to
activation
by
intact,
infected
erythrocytes
(iRBC)
or
freeze-thawed
schizont
lysate
(PfSL)
and
(iii)
determine
the
activation
requirements
for
innate
immune
cells
responding
to
P.
falciparum.
Infected
erythrocytes
induce
a
more
rapid
and
intense
IFN-y
response
from
malaria
naive
PBMC
than
does
PfSL,
correlating
with
rapid
iRBC
activation
of
CD3-CD56+
natural
killer
(NK)
cells
to
produce
IFN-y.
There
is
marked
heterogeneity
between
donors
in
the
magnitude
of
the
NK-IFN-y
response
not
correlating
with
mitogen
or
cytokine-induced
NK
activation
or
prior
malaria
exposure.
The
NK-IFN-y
response
is
highly
IL-I2
dependent,
partly
IL-I8
dependent
and
highly
dependent
on
direct
contact
between
the
NK
cell
and
the
parasitized
erythrocyte.
Exogenous
rIL-I2
or
rIL-I8
did
not
augment
NK-IFN-y
responses
indicating
that
IL-I2
and
IL-18
production
is
not
the
limiting
factor
explaining
differences
in
NK
cell
reactivity
between
live
and
dead
parasites
or
between
donors.
The
possibility
that
donor
heterogeneity
is
due
to
genetic
variation
in
killer
immunoglobulin-
like
receptors
(KIR)
and/or
differential
expression
of
C-type
lectin
receptors
was
also
investigated.
A
significant
up-regulation
ofCD94
and
NKG2A
was
observed
in
IFN-y+
NK
cells
of
responding
donors,
suggesting
that
the
inhibitory
CD94:NKG2A
heterodimer
may
serve
a
regulatory
function
on
P.
falciparum
activated
NK
cells.
Collectively,
these
data
indicate
that
NK
cells
may
represent
an
important
early
source
oflFN-y,
a
cytokine
implicated
in
induction
of
various
anti-parasitic
effector
mechanisms.
The
heterogeneity
of
this
early
IFN-y
response
between
donors
suggests
variation
in
their
ability
to
mount
a
rapid
pro-inflammatory
cytokine
response
to
malaria
that
may,
in
turn,
influence
their
innate
susceptibility
to
malaria
infection,
malaria-related
morbidity
or
death
from
malari
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Regulation of ligand-independent notch signal through intracellular trafficking
Notch signaling is an evolutionarily conserved mechanism that defines a key cell fate control mechanism in metazoans. Notch signaling relies on the surface interaction between the Notch receptor and membrane bound ligands in an apposing cell. In our recent study, we uncover a non-canonical receptor activation path that relies on a ligand-independent, intracellular activation of the receptor as it travels through the endosomal compartments. We found that Notch receptor, targeted for degradation lysosomal degradation through multivesicular bodies (MVBs) is “diverted” toward activation upon mono-ubiquitination through a synergy between the ubiquitin ligase Deltex, the non-visual β-arrestin Kurtz and the ESCRT-III component Shrub. This activation path is not universal but appears to depend on the cellular context
Down-regulation of Delta by proteolytic processing
Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner
An actin-related protein in Drosophila colocalizes with heterochromatin protein 1 in pericentric heterochromatin
The actin-related proteins have been identified by virtue of their sequence similarity to actin. While their structures are thought to be closely homologous to actin, they exhibit a far greater range of functional diversity. We have localized the Drosophila actin-related protein, Arp4, to the nucleus. It is most abundant during embryogenesis but is expressed at all developmental stages. Within the nucleus Arp4 is primarily localized to the centric heterochromatin. Polytene chromosome spreads indicate it is also present at much lower levels in numerous euchromatic bands. The only other protein in Drosophila reported to be primarily localized to centric heterochromatin in polytene nuclei is heterochromatin protein 1 (HP1), which genetic evidence has linked to heterochromatin-mediated gene silencing and alterations in chromatin structure. The relationship between Arp4 and heterochromatin protein 1 (HP1) was investigated by labeling embryos and larval tissues with antibodies to Arp4 and HP1. Arp4 and HP1 exhibit almost superimposable heterochromatin localization patterns, remain associated with the heterochromatin throughout prepupal development, and exhibit similar changes in localization during the cell cycle. Polytene chromosome spreads indicate that the set of euchromatic bands labeled by each antibody overlap but are not identical. Arp4 and HP1 in parallel undergo several shifts in their nuclear localization patterns during embryogenesis, shifts that correlate with developmental changes in nuclear functions. The significance of their colocalization was further tested by examining nuclei that express mutant forms of HP1. In these nuclei the localization patterns of HP1 and Arp4 are altered in parallel fashion. The morphological, developmental and genetic data suggest that, like HP1, Arp4 may have a role in heterochromatin functions. Keywords: Chromatin, Actin-related protein, Drosophila, Heterochromatin-protein 1, Position effect variegatio
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Drosophila Protein interaction Map (DPiM): A paradigm for metazoan protein complex interactions
Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein—especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex “map” provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map
Large-scale proteomic analysis of T. spiralis muscle-stage ESPs identifies a novel upstream motif for in silico prediction of secreted products
The Trichinella genus contains parasitic nematodes capable of infecting a wide range of hosts including mammals, birds and reptiles. Like other helminths, T. spiralis secretes a complex mixture of bioactive molecules capable of modulating its immediate surroundings and creating a hospitable environment for growth, survival and ultimately transmission. The constitution of these excretory-secretory products (ESPs) changes depending on the tissue niche and the specific stage of parasite development. Unique to T. spiralis is a true intracellular stage wherein larvae develop inside striated myotubes. Remarkably, the parasite larvae do not destroy the host cell but rather reprogram it to support their presence and growth. This transformation is largely mediated through stage-specific secretions released into the host cell cytoplasm. In this study, we apply state of the art proteomics and computational approaches to elucidate the composition and functions of muscle-stage T. spiralis ESPs. Moreover, we define a recurring, upstream motif associated with the stichosome, the main secretory organ of this worm, and can be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages
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Molecular Structure and Dimeric Organization of the Notch Extracellular Domain as Revealed by Electron Microscopy
Background: The Notch receptor links cell fate decisions of one cell to that of the immediate cellular neighbor. In humans, malfunction of Notch signaling results in diseases and congenital disorders. Structural information is essential for gaining insight into the mechanism of the receptor as well as for potentially interfering with its function for therapeutic purposes. Methodology/Principal Findings: We used the Affinity Grid approach to prepare specimens of the Notch extracellular domain (NECD) of the Drosophila Notch and human Notch1 receptors suitable for analysis by electron microscopy and three-dimensional (3D) image reconstruction. The resulting 3D density maps reveal that the NECD structure is conserved across species. We show that the NECD forms a dimer and adopts different yet defined conformations, and we identify the membrane-proximal region of the receptor and its ligand-binding site. Conclusions/Significance: Our results provide direct and unambiguous evidence that the NECD forms a dimer. Our studies further show that the NECD adopts at least three distinct conformations that are likely related to different functional states of the receptor. These findings open the way to now correlate mutations in the NECD with its oligomeric state and conformation
Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal
The ESCRT-III complex component Shrub plays a pivotal rate-limiting step in late endosomal ligand-independent Notch activation
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