110 research outputs found
Untersuchung der See-, Manövrier- und LeistungsfĂ€higkeit des Semi-Katamarans fĂŒr den Containertransport im durchgehenden Fluss-See-Verkehr
Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER
Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of
around 10,000 different soluble and membrane proteins in humans. It involves the co- or post translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion
or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four
pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver
their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the pre cursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which
may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes,
or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the
PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting vari ous peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act
in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either
remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components
of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more
general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting
of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent
pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by
an established approach which involved the label-free quantitative mass spectrometry of the total
proteome of depleted or deficient cells, as well as differential protein abundance analysis. The
negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the
ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells.
Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane
helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which
may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures
in the respective Zellweger patients
Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER
In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting
of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER
membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide
chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane
post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of
mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding
proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were
studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the
conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches
such as proximity-based ribosome profiling or quantitative proteomics were employed to address
the question of which precursors use certain pathways under physiological conditions. Here, we
combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free
quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-
involving precursors and to identify possible genetic interactions between the various targeting
pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and
critically discuss the pros and cons of this experimental approach
Is keV ion induced pattern formation on Si(001) caused by metal impurities?
We present ion beam erosion experiments performed in ultra high vacuum using
a differentially pumped ion source and taking care that the ion beam hits the
Si(001) sample only. Under these conditions no ion beam patterns form on Si for
angles below 45 degrees with respect to the global surface normal using 2 keV
Kr ions and fluences of 2 x 10^22 ions/m^2. In fact, the ion beam induces a
smoothening of preformed patterns. Simultaneous sputter deposition of stainless
steel in this angular range creates a variety of patterns, similar to those
previously ascribed to clean ion beam induced destabilization of the surface
profile. Only for grazing incidence with incident angles between 60 degrees and
83 degrees pronounced ion beam patterns form. It appears that the angular
dependent stability of Si(001) against pattern formation under clean ion beam
erosion conditions is related to the angular dependence of the sputtering
yield, and not primarily to a curvature dependent yield as invoked frequently
in continuum theory models.Comment: 15 pages, 7 figures. This is an author-created, un-copyedited version
of an article published in Nanotechnology. IOP Publishing Ltd is not
responsible for any errors or omissions in this version of the manuscript or
any version derived from i
Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting
Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals
Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting
Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals
Jesuit Theologian to Lecture at USD
The 2,2′:6′:2″-terpyridine ligand has literally shaped the coordination chemistry of transition metal complexes in a plethora of fields. Expansion of the ligand bite by amine functionalities between the pyridine units in the tridentate N,N’-dimethyl-N,N’-dipyridine-2-yl-pyridine-2,6-diamine ligand (ddpd) modifies the properties of corresponding transition metal complexes, comprising redox chemistry, molecular dynamics, magnetism and luminescence. The origins of these differences between ddpd and tpy complexes will be elucidated and comprehensively summarized with respect to first row transition metal complexes with d2–d10 electron configurations. Emerging applications of these ddpd complexes complementary to those of the well-known terpyridine ligand will be highlighted
Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import
In mammalian cells, oneâthird of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex facilitates ER import of most precursor polypeptides, the Sec61âassociated Sec62/Sec63 complex supports ER import in a substrateâspecific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivoâlike conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxyâterminal region of SP (Câregion) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocationâdisruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavyâchain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61âchannel inhibitor CAM741. Thus, the human Sec62/Sec63 complex may support Sec61âchannel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic aminoâterminal peptide of Sec61α or via recruitment of BiP and its interaction with the ERâlumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63âlinked polycystic liver disease
Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting
Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals
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