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1.
Yet another protein has been added to the crowd of players found at the ends of chromosomes. Known variously as PTOP, PIP1 or TINT1, this negative regulator of telomere length connects some of the key proteins already known to be present - TRF1, TIN2, POT1, and TRF2 - and adds even more complexity to telomere protein interactions. 相似文献
2.
Peter M Huelsmann Andreas D Hofmann Stefanie A Knoepfel Jasmin Popp Pia Rauch Francesca Di Giallonardo Christina Danke Eva Gueckel Axel Schambach Horst Wolff Karin J Metzner Christian Berens 《BMC biotechnology》2011,11(1):4
Background
Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.Results
When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)2R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.Conclusions
This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.3.
Knoepfel SA Salisch NC Huelsmann PM Rauch P Walter H Metzner KJ 《Journal of virology》2008,82(13):6536-6545
4.
Mark S. Springer Christian F. Guerrero-Juarez Matthias Huelsmann Matthew A. Collin Kerri Danil Michael R. McGowen Ji Won Oh Raul Ramos Michael Hiller Maksim V. Plikus John Gatesy 《Current biology : CB》2021,31(10):2124-2139.e3
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5.
Huelsmann S Hepper C Marchese D Knöll C Reuter R 《Development (Cambridge, England)》2006,133(15):2915-2924
In Drosophila embryos, macrophages originate from the cephalic mesoderm and perform a complex migration throughout the entire embryo. The molecular mechanisms regulating this cell migration remain largely unknown. We identified the Drosophila PDZ G-nucleotide exchange factor (PDZ-GEF) Dizzy as a component essential for normal macrophage migration. In mutants lacking Dizzy, macrophages have smaller cellular protrusions, and their migration is slowed down significantly. This phenotype appears to be cell-autonomous, as it is also observed in embryos with a dsRNA-induced reduction of dizzy function in macrophages. In a complementary fashion, macrophages overexpressing Dizzy are vastly extended and form very long protrusions. These cell shape changes depend on the function of the small GTPase Rap1: in rap1 mutants, Dizzy is unable to induce the large protrusions. Furthermore, forced expression of a dominant-active form of Rap1, but not of the wild-type form, induces similar cell shape changes as Dizzy does overexpression. These findings suggest that Dizzy acts through Rap1. We propose that integrin-dependent adhesion is a Rap1-mediated target of Dizzy activity: in integrin mutants, neither Dizzy nor Rap1 can induce cell shape changes in macrophages. These data provide the first link between a PDZ-GEF, the corresponding small GTPase and integrin-dependent cell adhesion during cell migration in embryonic development. 相似文献
6.
Markus P. Kummer Hiroko Maruyama Claudia Huelsmann Sandra Baches Sascha Weggen Edward H. Koo 《The Journal of biological chemistry》2009,284(4):2296-2306
The formation of insoluble cross β-sheet amyloid is pathologically
associated with disorders such as Alzheimer, Parkinson, and Huntington
diseases. One exception is the nonpathological amyloid derived from the
protein Pmel17 within melanosomes to generate melanin pigment. Here we show
that the formation of insoluble MαC intracellular fragments of Pmel17,
which are the direct precursors to Pmel17 amyloid, depends on a novel
juxtamembrane cleavage at amino acid position 583 between the furin-like
proprotein convertase cleavage site and the transmembrane domain. The
resulting Pmel17 C-terminal fragment is then processed by the
γ-secretase complex to release a short-lived intracellular domain
fragment. Thus, by analogy to the Notch receptor, we designate this cleavage
the S2 cleavage site, whereas γ-secretase mediates proteolysis at the
intramembrane S3 site. Substitutions or deletions at this S2 cleavage site,
the use of the metalloproteinase inhibitor TAPI-2, as well as small
interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17
reduced the formation of insoluble Pmel17 fragments. These results demonstrate
that the release of the Pmel17 ectodomain, which is critical for melanin
amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.Folding of proteins is a highly regulated process ensuring their correct
three-dimensional structure. Under pathological circumstances, a soluble
protein can be folded into highly stable cross β-sheet amyloid
structures, which are believed to play pathological roles in disorders such as
Alzheimer, Parkinson, and Huntington diseases. An exception to this general
concept is the physiological amyloid structure of the melanosomal matrix
formed by the protein Pmel17. Melanosomes are lysosome-related organelles that
contain pigment granules (melanin) in melanocytes and retinal epithelial cells
(reviewed in Ref. 1).
Melanogenesis is believed to proceed through several sequential maturation
steps, classified by melanosomes from stage I to stage IV. Maturation of stage
II melanosomes requires the formation of Pmel17 intralumenal fibers
(2,
3).Pmel17 (also called gp100, ME20, RPE1, or silver) is a type I transmembrane
glycoprotein of up to 668 amino acids in humans (reviewed in Ref.
4). The requirement of Pmel17
for the generation of functional melanin has been shown in a number of
different organisms, because, for example, certain point mutations in the
Pmel17/silver gene result in hypopigmentation phenotypes
(5–7).
The most characteristic domain within Pmel17 is a specific lumenal
proline/serine/threonine rich repeat domain (see
Fig. 1A), that is
imperfectly repeated 13 times in the Mα fragment. Importantly, deletion
of the rich repeat domain results in a complete loss of fibril formation,
pointing to the requirement of Pmel17, and especially the rich repeat domain,
in melanin formation (8).
Pmel17 exists in different isoforms generated by alternative splicing.
Pmel17-i2 is the most
abundant isoform, whereas the Pmel17-l isoform contains a 7-amino acid
insertion close to the transmembrane domain
(9,
10).Open in a separate windowFIGURE 1.Effect of the γ-secretase inhibitor DAPT on Pmel17 processing.
A, schematic diagram of Pmel17 and epitopes of antibodies. Pmel17
contains five potential N-glycosylation sites indicated by branched
structures. The long form of Pmel17, Pmel17-l, is characterized by a seven
amino acid insertion (VPGILLT) within the lumenal domain close to the
transmembrane domain (TM), which is absent in Pmel17-i. NVS marks a
potential N-glycosylation site near this insertion. The epitopes of
antibodies αPep13h and HMB45 are indicated. Cleavage by a furin-like PC
results in the formation of the Mα and the membrane-bound 26-kDa Mβ
fragment, which are connected via disulfide bonds. Release and further
processing of the Mα fragment into MαN and MαC fragments
results in the formation of fibrils and marks the transition of stage I to
stage II melanosomes (dashed line). B, human MNT-1 cells
were incubated with increasing amounts of DAPT for 18 h, and then the lysates
were separated by SDS-PAGE and analyzed by immunoblotting with αPep13h
antibody. DAPT treatment resulted in the accumulation of a C-terminal fragment
of Pmel17 (CTF), whereas Pmel17 P1 and Mβ fragment were unchanged.
C, probing the Triton-soluble fraction with HMB45 revealed increased
amounts of the highly glycosylated P2 form of Pmel17 after DAPT incubation.
D, detection of Pmel17 amyloidogenic fragments (MαC) in the
SDS-extracted insoluble pellet using antibody HMB45. E, murine B16-FO
cells treated with increasing concentrations of DAPT. Immunoblotting using
antibodyαPep13h revealed the formation of CTF of similar size as in
MNT-1 cells. F, time course analysis of Pmel17, Mβ, and
Pmel17-CTF after DAPT treatment. The cell lysates were immunoblotted using
αPep13h. Pmel17-CTF was detectable after 10 min of incubation with 1
μm DAPT. G, the size of the Pmel17-CTF was determined
using an unstained low molecular range peptide standard. The marker peptides
were detected by Ponceau S staining and Pmel17-CTF were detected by immunoblot
using αPep13h.Pmel17 traffics through the secretory pathway as a 100-kDa protein (called
P1). In the late Golgi compartment it undergoes further glycosylation,
resulting in a short lived 120-kDa protein (called P2). P2 is rapidly cleaved
within the post-Golgi by a furin-like proprotein convertase (PC) to generate
two fragments that remain tethered to each other by disulfide bonds: a
C-terminal polypeptide containing the transmembrane domain (Mβ) and a
large N-terminal ectodomain (Mα)
(2)
(Fig. 1A).
Consequently, inhibition of this furin-like activity not only prevents the
generation of Mα and Mβ fragments but also inhibits the formation
of melanosomal striation in HeLa cells
(3). These findings suggest
that Mα must first be dissociated from the Mβ for melanogenesis to
proceed. It is unclear how Mα is released from the membrane. Reduction
of disulfide bonds would release Mα from Mβ; alternatively,
proteolytic digestion of Mβ should also free Mα from the membrane
tether. It has been speculated that, given the presence of lysosomal
hydrolases in melanosomes and proteolytic maturation of Pmel17, proteolysis is
the more likely mechanism (4).
Recently, it was shown that recombinant Mα is able to form amyloid
structures in vitro in an unprecedented rapidity, and furthermore,
Pmel17 amyloid also accelerated melanin formation
(11). These findings
demonstrate that mammalian amyloid formed by Pmel17 is functional and
physiological.The insoluble pool of Pmel17 in cells consists mostly of truncated Mα
C-terminal fragments (MαC) of heterogeneous sizes, indicating that
further processing of Mα occurs after its release from the membrane
(8,
12). MαC fragments are
found in the insoluble fraction of melanocytes as well as in nonmelanotic
cells, the latter after overexpression of Pmel17
(8), and are reduced or absent
in amelanotic cells (8,
13,
14). Meanwhile, the C-terminal
fragment derived from the Mβ fragment and recognized by a C-terminal
specific epitope antibody is less stable, indicating rapid turnover
(2).The presenilin (PS) family of proteins consists of two homologous integral
transmembrane proteins, PS1 and PS2, which are part of the γ-secretase
complex. The latter consists of presenilin 1 or 2, nicastrin, APH-1, and PEN-2
(15) and catalyzes the
cleavage of the hydrophobic transmembrane domain of a burgeoning list of
proteins, also called regulated intramembrane cleavage. Other substrates for
the γ-secretase-mediated intramembrane cleavage include Notch, amyloid
precursor protein (APP), cadherin (E-cadherin), nectin-1, the low density
lipoprotein-related receptor, CD44, ErbB-4, the voltage-gated sodium channel
β2-subunit, and the Notch ligands Delta and Jagged. Importantly, in
Alzheimer disease, the presenilin-mediated γ-secretase cleavage of APP
releases the amyloid β-protein fragment, a peptide believed to play a key
role in Alzheimer disease pathogenesis. Interestingly, a recent report
described the absence of melanin pigment in presenilin-deficient animals, an
observation confirmed by the lack of melanin formation in cells treated with
γ-secretase inhibitors
(16). The mechanism
responsible for this finding is unclear, leading us to ask whether Pmel17
processing is a presenilin-dependent process and, if so, whether this cleavage
is involved in melanogenesis.In this study, we show the presence of an endoproteolytic activity that
cleaves the extracellular domain of Pmel17-i at a juxtamembrane position
between the known PC cleavage site and the transmembrane domain, which we term
the S2 cleavage site, by a TAPI-sensitive ADAM (a disintegrin
and metalloproteinase protein) protease. This
intracellular shedding of Pmel17 after S2 cleavage results in the liberation
of the Mα N-terminal ectodomain, the precursor to Pmel17 amyloid, which
is able to form insoluble Pmel17 aggregates. The C-terminal transmembrane
fragment generated by S2 cleavage is further processed by γ-secretase
(S3 cleavage) to release the Pmel17 intracellular domain, which is then
rapidly degraded. 相似文献
7.
Human POT1 facilitates telomere elongation by telomerase 总被引:39,自引:0,他引:39
Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase. 相似文献
8.
PH and growth of Torulopsis pintolopesii in media containing various sugars as carbon and energy sources. 总被引:1,自引:1,他引:0
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Growth within the pH range 2 to 8 of a strain of the yeast Torulopsis pintolopesii was tested in media containing various sugars as carbon and energy sources. Of the sugars tested, only D-glucose, D-fructose, and D-mannose supported growth of the yeast. In media containing those sugars, the organism grew over the entire pH range tested. 相似文献
9.
10.
SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype. 总被引:2,自引:0,他引:2
Christian Darimont Ornella Avanti Yvonne Tromvoukis Patricia Vautravers-Leone Nori Kurihara G David Roodman Lorel M Colgin Heide Tullberg-Reinert Andrea M A Pfeifer Elizabeth A Offord Katherine Mace 《Cell growth & differentiation》2002,13(2):59-67
In most human primary bone cells, SV40 T-antigen expression was able to expand life span for a few passages before cells undergo growth arrest, described as crisis. In this study, telomerase activity was reconstituted in human osteoblast precursors (hPOB cells) and marrow stromal cells (Saka cells) transformed with the SV40 T antigen. Bone cells with telomerase activity were able to bypass crisis and show unlimited life span. Despite chromosomal aberrations observed in hPOB-tert cells, these immortalized precursors were able to differentiate into osteoblasts like precrisis hPOB cells. Saka-tert cells enhanced the formation of human osteoclast-like cells in a similar manner as Saka cells. These results demonstrate that reconstitution of telomerase activity in transformed SV40 T-antigen human osteoblast precursors or marrow stromal cells leads to the generation of immortalized cells with a preserved phenotype. 相似文献