The subset of peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.

TSignal: a transformer model for signal peptide prediction.

MOTIVATION: Signal peptides (SPs) are short amino acid segments present at the N-terminus of newly synthesized proteins that facilitate protein translocation into the lumen of the endoplasmic reticulum, after which they are cleaved off. Specific regions of SPs influence the efficiency of protein translocation, and small changes in their primary structure can abolish protein secretion altogether. The lack of conserved motifs across SPs, sensitivity to mutations, and variability in the length of the peptides make SP prediction a challenging task that has been extensively pursued over the years. RESULTS: We introduce TSignal, a deep transformer-based neural network architecture that utilizes BERT language models and dot-product attention techniques. TSignal predicts the presence of SPs and the cleavage site between the SP and the translocated mature protein. We use common benchmark datasets and show competitive accuracy in terms of SP presence prediction and state-of-the-art accuracy in terms of cleavage site prediction for most of the SP types and organism groups. We further illustrate that our fully data-driven trained model identifies useful biological information on heterogeneous test sequences. AVAILABILITY AND IMPLEMENTATION: TSignal is available at: https://github.com/Dumitrescu-Alexandru/TSignal.

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins.

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature.

Mycolactone is a bacteria-derived macrolide that blocks the biogenesis of a large array of secretory and integral transmembrane proteins (TMP) through potent inhibition of the Sec61 translocon. Here, we used quantitative proteomics to delineate the direct and indirect effects of mycolactone-mediated Sec61 blockade in living cells. In T lymphocytes, dendritic cells and sensory neurons, Sec61 substrates downregulated by mycolactone were in order of incidence: secretory proteins (with a signal peptide but no transmembrane domain), TMPs with a signal peptide (Type I) and TMPs without signal peptide and a cytosolic N terminus (Type II). TMPs without a signal peptide and the opposite N terminus topology (Type III) were refractory to mycolactone inhibition. This rule applied comparably to single- and multi-pass TMPs, and extended to exogenous viral proteins. Parallel to its broad-spectrum inhibition of Sec61-mediated protein translocation, mycolactone rapidly induced cytosolic chaperones Hsp70/Hsp90. Moreover, it activated an atypical endoplasmic reticulum stress response, differing from conventional unfolded protein response by the down-regulation of Bip. In addition to refining our mechanistic understanding of Sec61 inhibition by mycolactone, our findings thus reveal that Sec61 blockade induces proteostatic stress in the cytosol and the endoplasmic reticulum.

Mycolactone subverts immunity by selectively blocking the Sec61 translocon.

Mycolactone, an immunosuppressive macrolide released by the human pathogen Mycobacterium ulcerans, was previously shown to impair Sec61-dependent protein translocation, but the underlying molecular mechanism was not identified. In this study, we show that mycolactone directly targets the α subunit of the Sec61 translocon to block the production of secreted and integral membrane proteins with high potency. We identify a single-amino acid mutation conferring resistance to mycolactone, which localizes its interaction site near the lumenal plug of Sec61α. Quantitative proteomics reveals that during T cell activation, mycolactone-mediated Sec61 blockade affects a selective subset of secretory proteins including key signal-transmitting receptors and adhesion molecules. Expression of mutant Sec61α in mycolactone-treated T cells rescued their homing potential and effector functions. Furthermore, when expressed in macrophages, the mycolactone-resistant mutant restored IFN-γ receptor-mediated antimicrobial responses. Thus, our data provide definitive genetic evidence that Sec61 is the host receptor mediating the diverse immunomodulatory effects of mycolactone and identify Sec61 as a novel regulator of immune cell functions.

Apratoxin Kills Cells by Direct Blockade of the Sec61 Protein Translocation Channel.

Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes.

Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding.

Synaptic adhesion molecules are key components in development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of chemical synapses. Several families of neuronal adhesion molecules have been identified such as the neuronal cell adhesion molecules, neurexins and neuroligins, and in particular recently several leucine-rich repeat proteins, e.g., Netrin G-ligands, SLITRKs, and LRRTMs. The LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic synapse function and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have been found to be ligands for heparan sulfate proteoglycans, including glypican. We report here the crystal structure of a thermostabilized mouse LRRTM2, with a Tm 30 °C higher than that of the wild-type protein. We localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation, and prior information about the interaction of the ligand with neurexins, which allowed us to propose a tentative model for the LRRTM-neurexin interaction complex. We also determined affinities of the thermostabilized LRRTM2 and wild-type LRRTM1 and LRRTM2 for neurexin-ß1 with and without Ca(2+). Cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapselike contacts. The structural and functional data presented here provide the first structure of an LRRTM protein and allow us to propose a model for the molecular mechanism of LRRTM function in the synaptic adhesion.

LRRTM3 is dispensable for amyloid-ß production in mice.

Neuronal LRRTM3 (leucine-rich repeat transmembrane 3) protein has been reported to promote amyloid-ß protein precursor (AßPP) processing and LRRTM3 is a candidate gene in late-onset Alzheimer's disease. To address the role of LRRTM3 in AßPP processing and amyloid-ß (Aß) production in vivo, we analyzed amyloidogenic processing of AßPP in the brains of LRRTM3-deficient mice and transgenic AßPP/PS1 mice with or without LRRTM3. We did not find differences between the genotypes in the levels of Aß or AßPP C-terminal fragments indicating that LRRTM3 is not an essential regulator of Aß production in adult mice. Moreover, Aß levels in primary cortical neurons were similar between the genotypes, indicating that LRRTM3 is not required for Aß generation in developing mice.

Bacteriophage Mu integration in yeast and mammalian genomes.

Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed 'transpososomes'. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.

Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage phi6 is required for single-stranded RNA packaging and transcription.

Bacteriophage phi6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.