Cross-linking of the Endolysosomal System Reveals Flotillin Structures and Putative Cargo

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes, but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyzed lysosomes and early endosomes. Based on the identification of 5,376 cross-links, we investigated protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and heterodimeric/-multimeric structures of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identified >300 proteins presenting putative cargo, and confirm the latrophilin family of adhesion G protein-coupled receptors as substrates for flotillin-dependent endocytosis.

The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer’s disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

Inhibition of Clostridioides difficile Toxins TcdA and TcdB by Ambroxol

Clostridioides (C.) difficile produces the exotoxins TcdA and TcdB, which are the predominant virulence factors causing C. difficile associated disease (CDAD). TcdA and TcdB bind to target cells and are internalized via receptor-mediated endocytosis. Translocation of the toxins’ enzyme subunits from early endosomes into the cytosol depends on acidification of endosomal vesicles, which is a prerequisite for the formation of transmembrane channels. The enzyme subunits of the toxins translocate into the cytosol via these channels where they are released after auto-proteolytic cleavage. Once in the cytosol, both toxins target small GTPases of the Rho/Ras-family and inactivate them by mono-glucosylation. This in turn interferes with actin-dependent processes and ultimately leads to the breakdown of the intestinal epithelial barrier and inflammation. So far, therapeutic approaches to treat CDAD are insufficient, since conventional antibiotic therapy does not target the bacterial protein toxins, which are the causative agents for the clinical symptoms. Thus, directly targeting the exotoxins represents a promising approach for the treatment of CDAD. Lately, it was shown that ambroxol (Ax) prevents acidification of intracellular organelles. Therefore, we investigated the effect of Ax on the cytotoxic activities of TcdA and TcdB. Ax significantly reduced toxin-induced morphological changes as well as the glucosylation of Rac1 upon intoxication with TcdA and TcdB. Most surprisingly, Ax, independent of its effects on endosomal acidification, decreased the toxins’ intracellular enzyme activity, which is mediated by a catalytic glucosyltransferase domain. Considering its undoubted safety profile, Ax might be taken into account as therapeutic option in the context of CDAD.

Inflammation in Focus: The Beginning and the End

The inflammation is an important biological response induced by various harmful stimuli, like viruses, bacterial infections, toxins, toxic compounds, tissue injury. During inflammation inflammatory cytokines and reactive oxygen species are produced. Inflammatory cytokines act on various receptors present on the plasma membrane of target cells. To initiate signaling cascade, and activate transcription factors, receptors should be internalized and enter the early endosomes, where the members of the signaling cascade can meet. The further cytoplasmic fate of the receptor plays crucial role in the progression and the course of inflammation. Usually acute inflammation removes injurious stimuli and helps to regain the normal healthy status of the organism. In contrast to this the uncontrolled chronic inflammation—stimulating other than immune cells, inducing transdifferentiation—can provide base of various serious diseases. This paper draws the attention of the long-lasting consequence of chronic inflammation, pointing out that one of the most important step in medication is to identify in time the factors initiating and maintaining inflammation.

Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β and γ secretases leads to the generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aβ42:Aβ40 ratio and the accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations—M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS-Hook-FHIP complexes

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS-Hook-FHIP ('FHF') cargo adaptor complex links dynein to cargo in mammals and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein 'interactome' of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 co-localize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS-Hook-FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in pathogenesis of Azheimer’s disease (AD). Sequential cleavage of APP by β and γ secretases leads to generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) pay a role of catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in increased Aβ42:Aβ40 ratio and accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study we performed molecular modeling of APP complex with γ-secretase and analyzed potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures we proposed that APP can form a complex with γ-secretase in 2 potential conformations – M1 and M2. In conformation M1 transmembrane domain of APP forms a contact with perimembrane domain that follows the transmembrane domain 6 (TM6) in PS1 structure. In conformation M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in PS1 structure. By analyzing effects of PS1-FAD mutations on local protein disorder index, we discovered that these mutations increase conformational flexibility of M2 and reduce conformational flexibility of M1. Based on these results we proposed that M2 conformation, but not M1 conformation, of γ secretase complex with APP leads to amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by a curved membranes, such as membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by a relatively flat memranes such as membranes of late endosomes and plasma membrane. These predictions are consistent with published biochemical analysis of APP processing at different subcellular locations. Our results suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting M2 conformation of γ secretase complex with APP.

Primaquine Inhibits the Endosomal Trafficking and Nuclear Localization of EGFR and Induces the Apoptosis of Breast Cancer Cells by Nuclear EGFR/Stat3-Mediated c-Myc Downregulation

Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.