Characterization of the transmembrane protein 109 in SRP-independent protein targeting to the human endoplasmic reticulum and its involvement in the cellular calcium homeostasis

Protein transport to the endoplasmic reticulum mainly relies on the signal recognition particle pathway. However, in the past decades, novel pathways and modes of protein transport have been identified to complement known transport routes for specific protein substrates to reach the endoplasmic reticulum. The signal recognition particle-independent (SND) pathway was identified in yeast cells as a tripartite system (Snd1-3). It presumably conducts protein clients to the endoplasmic reticulum due to a targeting signal located downstream of the N-terminus. However, the entire pathway has not been identified in human cells, so far. Only one component, the ER membrane protein hSnd2, is known in mammalian cells. This work aimed to identify the missing ER membrane protein, the hSnd3 of the pathway. Previous co-immunoprecipitation and subsequent mass spectrometry results revealed the interacting proteins with the hSnd2. Amongst these proteins, the transmembrane protein 109 (TMEM109) was enlisted, and it also showed simultaneous lower abundance when hSnd2 was knocked down. TMEM109 is a three-transmembrane domain protein resident in the endoplasmic reticulum membrane. A luminescence reporter assay described in this work also confirmed the interaction between the two proteins in living cells. TMEM109 knockdown resulted in the compensatory elevated abundance of the translocon Sec61α subunit and known targeting receptors, such as the signal recognition particle receptor. Accordingly, an improved transport efficiency of signal recognition particle-dependent substrates was observed. These results were consistent and congruent with data obtained when hSnd2 was knocked down. The model protein, cytochrome b5, a tail-anchored membrane protein showed partial dependence on the SND pathway based on in vitro translation assay results; however, re-expressing TMEM109 after its silencing did not rescue its transport efficiency to the endoplasmic reticulum. Using the CRISPR-Cas9 gene-editing system, a TMEM109 knockout cell line was generated. In TMEM109 knockout cells, similar but much more pronounced compensatory effects as in the knockdown cells were observed. Conversely, the partial SND dependence of the tail-anchored membrane protein, cytochrome b5 was not observed in TMEM109 knockout cells, likely due to the activated compensatory mechanisms. TMEM109 has been described as a calcium channel in the endoplasmic reticulum membrane. Live-cell calcium imaging shed light on the fact that lower TMEM109 abundance can disturb the calcium homeostasis; however, it was not observed in the cells completely lacking TMEM109. Based on the obtained results, TMEM109 is possibly implemented in protein targeting to the endoplasmic reticulum as an SND pathway element in human cells. However, no bona fide human SND pathway substrate has been identified so far. Further research would address the subset of proteins that require the SND pathway to transport to the endoplasmic reticulum. It is also possible that the SND pathway complements already known transport pathways, or it is activated under specific cellular circumstances.Der Proteintransport zum endoplasmatischen Retikulum beruht hauptsächlich auf dem Signalerkennungspartikelweg. In den letzten Jahrzehnten wurden jedoch neue Wege und Arten des Proteintransports identifiziert, die zum endoplasmatischen Retikulum bekannte Transportwege für bestimmte Proteinsubstrate ergänzen oder ersetzen. Der Signalerkennungspartikel-unabhängige (SND) Weg wurde in Hefezellen als Dreikoponentensystem (Snd1-3) identifiziert und leitet aufgrund eines Zielsignals Proteine zu dem endoplasmatischen Retikulum, welches sich vermutlich abseits des N-Terminus befindet. In menschlichen Zellen wurde bisher nicht der gesamte Signalweg identifiziert. Nur eine Komponente, das ER-Membranprotein hSnd2, ist in Säugetierzellen bekannt. Das Ziel dieser Arbeit war das fehlende ER-Membranprotein, hSnd3 des Weges zu identifizieren. Frühere Co-Immunopräzipitation und anschließende Massenspektrometrie-Ergebnisse zeigten die mit hSnd2 interagierenden Proteine. Es wurde unter anderem das Transmembranprotein 109 (TMEM109) identifiziert, welches zudem eine verringerte Abundanz nach siRNA-vermittelter hSnd2 Depletion aufwies. TMEM109 ist ein Membranprotein des endoplasmatischen Retikulums mit drei Transmembrandomänen. Ein Lumineszenz-Reporter-Assay, der in dieser Arbeit beschrieben wurde, bestätigte ebenfalls die Interaktion zwischen den beiden Proteinen in lebenden Zellen. Der Knockdown von TMEM109 führte zu einer kompensatorisch erhöhten Abundanz der zentralen Translokon-Untereinheit Sec61α und bekannter Targeting-Rezeptoren, wie etwa der Signalerkennungspartikel-Rezeptor. Dementsprechend wurde eine verbesserte Transporteffizienz von Signalerkennungspartikel-abhängigen Substraten beobachtet. Diese Ergebnisse stimmten mit den Daten überein, die durch Silencing von hSnd2 gewonnen wurden. Das Modellprotein Cytochrom b5, ein C-terminal verankerte Membranprotein zeigte eine partielle Abhängigkeit vom SND-Weg, basierend auf In-vitro-Translationsassay-Ergebnissen. Die Reexpression von TMEM109 nach Silencing rettete jedoch nicht seine Transporteffizienz zum endoplasmatischen Retikulum. Mithilfe des CRISPR-Cas9-Gen-Editing-Systems wurde eine TMEM109-Knockout-Zelllinie erzeugt. In TMEM109-Knockout-Zellen wurden zu den Knockdown-Proben ähnliche, aber deutlich stärkere, kompensatorische Änderungen beobachtet. Auf Grund der starken kompensatorischen Anpassung konnte die partielle SND-Abhängigkeit des C-terminal verankerten Membranproteins Cytochrom b5 in TMEM109-Knockout-Zellen nicht beobachtet werden. TMEM109 wurde als Kalziumkanal in der Membran des endoplasmatischen Retikulums beschrieben. Live-Cell-Calcium-Imaging hat gezeigt, dass eine geringere TMEM109-Abundanz die Kalzium-Homöostase stören kann. Dies konnte jedoch in den Zellen in denen TMEM109 vollständig fehlt, nicht beobachtet werden. Anhand dieser Ergebnisse nimmt TMEM109 in menschlichen Zellen möglicherweise als Element des SND-Wegs am Transport von Proteinen in das endoplasmatische Retikulum teil. Bisher wurde kein spezifisches Substrat des SND-Weges identifiziert. Weitere Forschungen würden sich mit solchen Proteine befassen, die den SND-Weg für ihren Transport zum endoplasmatischen Retikulum benötigen. Es ist auch möglich, dass der SND-Weg entweder bereits bekannte Transportwege ergänzt oder unter bestimmten zellulären Umständen aktiviert wird

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and 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

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

The 3q Oncogene SEC62 Predicts Response to Neoadjuvant Chemotherapy and Regulates Tumor Cell Migration in Triple Negative Breast Cancer

In the absence of targeted treatment options, neoadjuvant chemotherapy (NACT) is applied widely for triple-negative breast cancer (TNBC). Response to NACT is an important parameter predictive of oncological outcomes (progression-free and overall survival). An approach to the evaluation of predictive markers enabling therapy individualization is the identification of tumor driver genetic mutations. This study was conducted to investigate the role of SEC62, harbored at 3q26 and identified as a driver of breast cancer pathogenesis, in TNBC. We analyzed SEC62 expression in The Cancer Genome Atlas database, and immunohistologically investigated SEC62 expression in pre- and post-NACT tissue samples from 64 patients with TNBC treated at the Department of Gynecology and Obstetrics/Saarland University Hospital/Homburg between January 2010 and December 2018 and compared the effect of SEC62 on tumor cell migration and proliferation in functional assays. SEC62 expression dynamics correlated positively with the response to NACT (p ≤ 0.01) and oncological outcomes (p ≤ 0.01). SEC62 expression stimulated tumor cell migration (p ≤ 0.01). The study findings indicate that SEC62 is overexpressed in TNBC and serves as a predictive marker for the response to NACT, a prognostic marker for oncological outcomes, and a migration-stimulating oncogene in TNBC

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease

The janus face of death receptor signaling during tumor immunoediting

Cancer immune surveillance is essential for the inhibition of carcinogenesis. Malignantly transformed cells can be recognized by both the innate and adaptive immune systems through different mechanisms. Immune effector cells induce extrinsic cell death in the identified tumor cells by expressing death ligand cytokines of the tumor necrosis factor ligand family. However, some tumor cells can escape immune elimination and progress. Acquisition of resistance to the death ligand-induced apoptotic pathway can be obtained through cleavage of effector cell expressed death ligands into a poorly active form, mutations or silencing of the death receptors, or overexpression of decoy receptors and pro-survival proteins. Although the immune system is highly effective in the elimination of malignantly transformed cells, abnormal/dysfunctional death ligand signaling curbs its cytotoxicity. Moreover, DRs can also transmit pro-survival and pro-migratory signals. Consequently, dysfunctional death receptor-mediated apoptosis/necroptosis signaling does not only give a passive resistance against cell death but actively drives tumor cell motility, invasion, and contributes to consequent metastasis. This dual contribution of the death receptor signaling in both the early, elimination phase, and then in the late, escape phase of the tumor immunoediting process is discussed in this review. Death receptor agonists still hold potential for cancer therapy since they can execute the tumor-eliminating immune effector function even in the absence of activation of the immune system against the tumor. The opportunities and challenges of developing death receptor agonists into effective cancer therapeutics are also discussed