Ville Oskari Paavilainen

Coordinating expression of diverse secreted and membrane proteins poses a large challenge to cells, with initial insertion into the secretory pathway at the endoplasmic reticulum (ER) being the main bottleneck in mammalian expression systems. Diverse hydrophobic signal peptides guide nascent polypeptides into the secretory pathway with varying efficiency. However, identification of optimal signal peptide sequences that facilitate efficient protein production in a protein-specific manner remains a major challenge. Here, we present a new platform for efficient high-throughput identification of highly optimized signal peptides for secretory protein production in mammalian cells. The pooled approach enabled simultaneous testing of thousands of signal peptide sequences and identification of the ones that drive functional and efficient production of the protein of interest. We demonstrate the general applicability of the platform using different biotechnological protein classes. The resulting sequence data combined with functional knowledge can enable future machine learning approaches for prediction of efficient signal peptides as well as de novo generation of new synthetic signal peptides for modular control of protein biogenesis.

In plant development, receptor kinases are often active in disparate cell types, with each requiring vastly different signaling outputs. The ERECTA (ER) receptor kinase and its homologs Erecta-Like1 (ERL1) and ERL2 exemplify this pleiotropy. In Arabidopsis , they influence stomatal patterning, shoot meristem function, and vascular cambium activity, among other functions. Such diverse functionality raises the question of how ER signaling can specify distinct cell behaviors. One key mechanism occurs via cell-type specific interactions with coreceptors, ligands, or other proteins that modulate signaling. However, little is known about ER interactors in the vascular cambium, a bifacial stem cell niche that generates phloem and xylem. Combinatorial mutations between ER , ERL1, and ERL2 and receptor kinases of a second family, Phloem Intercalated with Xylem ( PXY) , PXY-LIKE1 ( PXL1) , and PXL2 , show severe cambial defects, but the mechanism underpinning these phenotypes is not known. Here, we show that PXY and PXL proteins form protein complexes with ER family members. In genetic analyses, plant lines in which PXY signaling was constitutively active had dramatic phenotypic changes that required the presence of ER or ERL2. Our results demonstrate that PXY signaling mediated cambium regulation in part depends on ER signaling and explains ER function in the cambium. Because the cambium produces xylem, which constitutes the wood in vascular plants, our findings position PXY–ER complexes at the center of the accumulation of this versatile biomaterial and essential carbon sink.

The genetically authenticated Finnish hop genotype LUKE 2541 obtained from wild was evaluated for antibacterial, anti-inflammatory, and anticancer activities. Water extracts from hop cones inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus , with MIC values of 0.094– 0.188⍰mg/mL, whereas Gram-negative strains showed limited sensitivity. In LPS-primed THP-1 cells, both IPA and IPA-Control extracts reduced reactive oxygen species formation in a dose-dependent manner, exhibiting similar IC ₅₀ values (50.41⍰µg/mL and 35.41⍰µg/mL). This hop genotype also displayed clear tissue- and solvent-dependent antiproliferative effects in human cancer cell lines. Bioactivity was strongly enriched in hop cones and predominantly associated with non-polar extracts, particularly hexane and dichloromethane fractions, which produced marked, dose-dependent reductions in cell viability. In contrast, aqueous and methanolic extracts were largely inactive, underscoring the critical importance of extraction chemistry and tissue selection. Sensitivity varied among cancer cell lines, with colorectal cells generally more responsive and leukemia cells less affected, highlighting cell-specific susceptibility. Further research is needed to elucidate underlying mechanisms, determine selectivity toward non-malignant cells, and identify the active compounds responsible for all in all investigated effects.

Polyamines are often associated with ribosomes and are thought to stabilize their integrity. In Arabidopsis , the polyamine thermospermine (tSpm) affects xylem cell fate. tSpm induces translation of SUPPRESSOR-OF-ACAULIS51 (SAC51) and SAC51-LIKEs (SACLs), which inhibit heterodimerization of the xylem development proteins LONESOME-HIGHWAY (LHW) and TARGET-OF-MONOPTEROS5. Here, we report a methyltransferase, OVERACHIEVER, that methylates the peptidyl transferase center of the 25 S ribosomal RNA (rRNA). Residue m ³ U2952 promotes functional tSpm binding to a specific site connecting the P-site transfer RNA (tRNA) with rRNA residues in the peptidyl transferase center. This interaction enhances the translation of SACLs but inhibits that of LHW. Our study uncovers the dependency between a conserved rRNA base methylation and a polyamine in orchestrating cell fate decisions, highlighting a role for the ribosome chemical landscape in translational regulation.

Insect-associated fungi are prolific producers of peptidic natural products with potent biological activities. Paraisaria is a genus of insect-pathogenic fungi (Family Ophiocordycipitaceae) in the Order Hypocreales, which also includes other Cordyceps-like fungi. Such fungi are increasingly used as functional foods and nutraceuticals, but Paraisaria may also be unintentionally consumed through infected, potentially toxic, edible insects. Here we report the discovery, isolation, and structure elucidation of paraisariamides A-D (1-4) from Paraisaria cascadensis and paraisariamides E-H (5-8) from P. insignis. Total synthesis of 1 and 5-8 was instrumental for comprehensive structure assignment and provided pure compounds for advanced biological testing. The paraisariamide family of N-methylated cyclic heptapeptides has been detected in all Paraisaria specimens analyzed to date from a variety of ecological niches. Paraisariamides A-H (1-8) display differential cell-type specific toxicity to human cancer cells, and we demonstrate that the most cytotoxic paraisariamides E-H (5-8) potently and rapidly inhibit mammalian protein synthesis. Molecular cartography was used to visualize the spatial distribution of paraisariamides in a lyophilized specimen of a beetle larva parasitized by P. insignis. Localization of paraisariamides to the fungal endosclerotium within the host larva is consistent with a role in localized disruption of host protein synthesis.

Insect-associated fungi are prolific producers of peptidic natural products with potent biological activities. Paraisaria is a genus of insect-pathogenic fungi (Family Ophiocordycipitaceae) in the Order Hypocreales, which also includes other Cordyceps-like fungi. Such fungi are increasingly used as functional foods and nutraceuticals, but Paraisaria may also be unintentionally consumed through infected, potentially toxic, edible insects. Here we report the discovery, isolation, structure elucidation and total synthesis of paraisariamides A-H, members of a new family of N-methylated cyclic heptapeptides detected in all Paraisaria specimens analyzed to date from a variety of ecological niches. Paraisariamides A-H (1-8) display differential, cell-type specific, toxicity to human cancer cells, and we demonstrate that the most cytotoxic paraisariamides E-H (5-8) potently and rapidly inhibit mammalian protein synthesis. Molecular cartography was used to visualize the spatial distribution of paraisariamides in a lyophilized specimen of a beetle larva parasitized by P. insignis. Localization of paraisariamides to the fungal endosclerotium within the host larva is consistent with a role in localized disruption of host protein synthesis.

Cellular homeostasis depends on the rapid, ATP-independent translocation of newly synthesized lipids across the endoplasmic reticulum (ER) membrane. Lipid translocation is facilitated by membrane proteins known as scramblases, a few of which have recently been identified in the ER. Our previous structure of the translocon-associated protein (TRAP) bound to the Sec61 translocation channel revealed local membrane thinning, suggesting that the Sec61/TRAP complex might be involved in lipid scrambling. Using complementary fluorescence spectroscopy assays, we detected nonselective scrambling by reconstituted translocon complexes. This activity was unaffected by Sec61 inhibitors that block its lateral gate, suggesting a second lipid scrambling pathway within the complex. Molecular dynamics simulations indicate that the trimeric TRAP subunit forms this alternative route, facilitating lipid translocation via a "credit card" mechanism, using a crevice lined with polar residues to shield lipid head groups from the hydrophobic membrane interior. Kinetic and thermodynamic analyses confirmed that local membrane thinning enhances scrambling efficiency and that both Sec61 and TRAP scramble phosphatidylcholine faster than phosphatidylethanolamine and phosphatidylserine, reflecting the intrinsic lipid flip-flop tendencies of these lipid species. As the Sec61 scrambling site lies in the lateral gate region, it is likely inaccessible during protein translocation, in line with our experiments on Sec61-inhibited samples. Hence, our findings suggest that the metazoan-specific trimeric TRAP bundle is a viable candidate for lipid scrambling activity that is insensitive to the functional state of the translocon.

Coibamide A (CbA) is a cyanobacterial lariat depsipeptide that selectively inhibits multiple secreted and integral membrane proteins from entering the endoplasmic reticulum secretory pathway through binding the alpha subunit of the Sec61 translocon. As a complex peptide-based macrocycle with 13 stereogenic centers, CbA is presumed to adopt a conformationally restricted orientation in the ligand-bound state, resulting in potent antitumor and antiangiogenic bioactivity. A stereochemical structure-activity relationship for CbA was previously defined based on cytotoxicity against established cancer cell lines. However, the ability of synthetic isomers to inhibit the biosynthesis of specific Sec61 substrates was unknown. Here, we report that two less toxic diastereomers of CbA, [L-Hiv²]-CbA and [L-Hiv², L-MeAla¹¹]-CbA, are pharmacologically active Sec61 inhibitors. Both compounds inhibited the expression of a secreted reporter (Gaussia luciferase), VEGF-A, and a Type 1 membrane protein (VCAM1), while [L-Hiv²]-CbA also decreased the expression of ICAM1 and BiP/GRP78. Analysis of 43 different chemokines in the secretome of SF-268 glioblastoma cells revealed different inhibitory profiles for the two diastereomers. When the cytotoxic potential of CbA compounds was compared against a panel of patient-derived glioblastoma stem-like cells (GSCs), Sec61 inhibitors were remarkably toxic to five of the six GSCs tested. Each ligand showed a distinct cytotoxic potency and selectivity pattern for CbA-sensitive GSCs, with IC₅₀ values ranging from subnanomolar to low micromolar concentrations. Together, these findings highlight the extreme sensitivity of GSCs to Sec61 modulation and the importance of ligand stereochemistry in determining the spectrum of inhibited Sec61 client proteins.