Retracing the evolution from antimicrobial to targeting peptides

We experimentally challenged the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert such antimicrobial peptides into bona fide organelle-targeting peptides, we expressed a set of 13 antimicrobial peptides of various origins in the green alga Chlamydomonas reinhardtii that serves as a model for both mitochondrial and chloroplast import. The peptides were modified to match distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. We present a temporal evolutionary scenario that emphasizes the early contribution of exchanging Lysines with Arginines in the sequence of the antimicrobial peptide, the evolution of a processing site followed by the addition of unstructured sequence and protein interaction sites that allow the selective targeting to the chloroplast.

CD47 interactions with exportin-1 limit the targeting of m7G-modified RNAs to extracellular vesicles

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5’-7-methylguanosine (m7G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m7G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m7G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m7G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m7G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex.

Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.

The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based ‘organelles’ found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25–50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure–function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier—it also plays a significant role in the structure and function of the cargo enzyme.

Cargo proteins in extracellular vesicles: potential for novel therapeutics in non-alcoholic steatohepatitis

Background Extracellular vesicles (EVs) are recognized as novel cell-free therapeutics. Non-alcoholic steatohepatitis (NASH) remains a critical health problem. Herein, we show that EVs from pan peroxisome proliferator-activated receptor agonist-primed induced mesenchymal stem cell (pan PPAR-iMSC-EVs) has unique cargo protein signatures, and demonstrate its therapeutic function in NASH. Results A unique protein signatures were identified in pan PPAR-iMSC-EVs against those from non-stimulated iMSC-EVs. NASH mice receiving pan PPAR-iMSC-EVs showed reduced steatotic changes and ameliorated ER stress and mitochondiral oxidative stress induced by inflammation. Moreover, pan PPAR-iMSC-EVs promoted liver regeneration via inhibiting apoptosis and enhancing proliferation. Conclusions We conclude that our strategy for enriching unique cargo proteins in EVs may facilitate the development of novel therapeutic option for NASH. Graphical Abstract

Encapsulin Based Self-Assembling Iron-Containing Protein Nanoparticles for Stem Cells MRI Visualization

Over the past decade, cell therapy has found many applications in the treatment of different diseases. Some of the cells already used in clinical practice include stem cells and CAR-T cells. Compared with traditional drugs, living cells are much more complicated systems that must be strictly controlled to avoid undesirable migration, differentiation, or proliferation. One of the approaches used to prevent such side effects involves monitoring cell distribution in the human body by any noninvasive technique, such as magnetic resonance imaging (MRI). Long-term tracking of stem cells with artificial magnetic labels, such as magnetic nanoparticles, is quite problematic because such labels can affect the metabolic process and cell viability. Additionally, the concentration of exogenous labels will decrease during cell division, leading to a corresponding decrease in signal intensity. In the current work, we present a new type of genetically encoded label based on encapsulin from Myxococcus xanthus bacteria, stably expressed in human mesenchymal stem cells (MSCs) and coexpressed with ferroxidase as a cargo protein for nanoparticles’ synthesis inside encapsulin shells. mZip14 protein was expressed for the enhancement of iron transport into the cell. Together, these three proteins led to the synthesis of iron-containing nanoparticles in mesenchymal stem cells—without affecting cell viability—and increased contrast properties of MSCs in MRI.

N -glycosylation of a cargo protein C-terminal domain recognized by the type IX secretion system in Cytophaga hutchinsonii affects protein secretion and localization

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes . It digests crystalline cellulose with an unknown mechanism, and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with CTD from CHU_2708. CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTD CHU_2708 fusion protein was found to be glycosylated in the periplasm with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide- N -glycosidase F which can hydrolyze N -linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTD CHU_2708 suggest that N -glycosylation occurred on the CTD. CTD N- glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii . Glycosyltransferase encoding gene chu_3842 , a homologous gene of Campylobacter jejuni pglA , was found to participate in the N -glycosylation of C. hutchinsonii . Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided the evidence that CTD as the signal of T9SS was N -glycosylated in the periplasm of C. hutchinsonii . IMPORTANCE The bacterial N -glycosylation system has previously only been found in several species of Proteobacteria and Campylobacterota , and the role of N -linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell-contact cellulose degradation mechanism, and many cell surface proteins including cellulases are secreted by the T9SS. Here, we found that C. hutchinsonii , a member of the phylum Bacteroidetes , has an N -glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N -glycosylation of C. hutchinsonii proteins, and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N -glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of CTD apears to play an important role in affecting T9SS substrates transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N -glycosylation in bacteria.

THADA drives Golgi residency and upregulation of PD-L1 in cancer cells and provides promising target for immunotherapy

BackgroundThe abnormal upregulation of programmed death-ligand 1 (PD-L1) in cancer cells inhibits T cell-mediated cytotoxicity, but the molecular mechanisms that drive and maintain PD-L1 expression are still incompletely understood.MethodsCombined analyses of genomes and proteomics were applied to find potential regulators of PD-L1. In vitro experiments were performed to investigate the regulatory mechanism of PD-L1 by thyroid adenoma associated gene (THADA) using human colorectal cancer (CRC) cells. The prevalence of THADA was analyzed using CRC tissue microarrays by immunohistochemistry. T cell killing assay, programmed cell death 1 binding assay and MC38 transplanted tumor models in C57BL/6 mice were developed to investigate the antitumor effect of THADA.ResultsTHADA is critically required for the Golgi residency of PD-L1, and this non-redundant, coat protein complex II (COPII)-associated mechanism maintains PD-L1 expression in tumor cells. THADA mediated the interaction between PD-L1 as a cargo protein with SEC24A, a module on the COPII trafficking vesicle. Silencing THADA caused absence and endoplasmic reticulum (ER) retention of PD-L1 but not major histocompatibility complex-I, inducing PD-L1 clearance through ER-associated degradation. Targeting THADA substantially enhanced T cell-mediated cytotoxicity, and increased CD8+ T cells infiltration in mouse tumor tissues. Analysis on clinical tissue samples supported a potential role of THADA in upregulating PD-L1 expression in cancer.ConclusionsOur data reveal a crucial cellular process for PD-L1 maturation and maintenance in tumor cells, and highlight THADA as a promising target for overcoming PD-L1-dependent immune evasion.

Structural and calorimetric studies reveal specific determinants for the binding of a high-affinity NLS to mammalian importin-alpha

The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its Nuclear Localization Sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3 the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.

Rabies Virus-Induced Autophagy Is Dependent on Viral Load in BV2 Cells

An increasing number of studies are showing that autophagy plays a vital role in viral replication and escape. Rabies virus (RABV), a typical neurotropic virus, has been proven to induce autophagy in neurons. However, there are no reports indicating that RABV can cause autophagy in other cells of the central nervous system. Thus, we aimed to explore the relationship between autophagy and RABV infection in BV2 cells in this study. Results of viral growth curves showed that the titers of microglial BV2 cells infected with RABV peaked at 12 hours post-infection (hpi) and then decreased continuously over time. However, it was found that the viral genome RNA and structural proteins can express normally in BV2 cells. In addition, Western blotting indicated that RABV infection increased LC3-II and p62 expression in BV2 cells. LC3 punctate increased with RABV infection in BV2 cells after the transfection of fluorescent protein-tagged LC3 plasmids. Moreover, autophagy cargo protein further accumulated with RABV infection in Bafilomycin A1-treated cells. Subsequently, RABV infection inhibited the fusion of autophagosomes with lysosomes by using a tandem fluorescent marker. Furthermore, a higher multiplicity of infection induced stronger autophagy. Thus, RABV can induce autophagy in BV2 cells, and the autophagy is positively associated with the viral load.