The vacuolar protein sorting genes in insects: A comparative genome view

Li, Zhaofei; Blissard, Gary W.

doi:10.1016/j.ibmb.2014.11.007

“…The vacuolar protein sorting (VPS) proteins perform this task with functions including protein recognition and recruitment to specific vesicular compartments, vesicle formation, vesicle transport, tethering of vesicles to a target membrane, and fusion with a target membrane. Subsets of VPS proteins form multiple functional complexes which include: endosomal sorting complex required for transport (ESCRT), Vps-C, Retromer, GARP, and PI3K sub-complex (Li and Blissard, 2015). The ESCRT machinery of eukaryotic cells is involved primarily in endosomal sorting and trafficking of cargo proteins to multivesicular bodies for protein degradation or autophagy.…”

Section: Resultsmentioning

confidence: 99%

“…That these protein trafficking components displayed such high sequence conservation is consistent with their central roles in many critical biological processes. Details of specific amino acid sequence and domain conservation and phylogeny of the VPS proteins are provided in a companion publication (Li and Blissard, 2015). In comparison with the yeast genome, VPS gene families are expanded in metazoans.…”

Section: Resultsmentioning

confidence: 99%

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

Kanost

¹

,

Arrese

²

,

Cao

³

et al. 2016

Insect Biochemistry and Molecular Biology

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Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

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“…One gene transcript, when depleted, strongly inhibited the cellular uptake and cytosolic delivery of both CPMP 2 R and stapled peptide 3 R : the VPS39 gene, also known as hVamp6. VPS39 encodes an 875-residue protein that is conserved in eukaryotes (55) and is one of six protein subunits comprising the HOPS membrane-tethering complex (SI Appendix, Fig. S7C).…”

Section: Sirna Depletion Of Hops But Not Corvet Inhibits the Cytosolicmentioning

confidence: 99%

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Steinauer

¹

,

LaRochelle

²

,

Knox

³

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cellbased assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7 + and Lamp1 + endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1 + late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.cell-penetrating peptides | peptidomimetics | enzyme replacement therapy | endocytosis | protein therapeutics

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“…One gene transcript, when depleted, strongly inhibited the cellular uptake and cytosolic delivery of both CPMP 2 R and stapled peptide 3 R : the VPS39 gene, also known as hVamp6. VPS39 encodes an 875-residue protein that is conserved in eukaryotes (68) and is one of six protein subunits comprising the homotypic fusion and vacuole protein sorting (HOPS) membrane tethering complex ( Figure 4A). Working with VPS41 and various effector proteins (69,70), VPS39 recruits the HOPS complex to endosomes positive for the small GTPase Rab7, such as maturing (Rab5 + /Rab7 + ) and late (Rab7 + ) endosomes.…”

Section: Sirna Depletion Of Hops But Not Corvet Inhibits the Cytosolimentioning

confidence: 99%

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Steinauer

¹

,

LaRochelle

²

,

Wissner

³

et al. 2018

Preprint

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Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7 + and Lamp1 + endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs-an environment that serves as a prerequisite for efficient endosomal escape. 4 conclude that HOPS allows CPMPs to traffic into intraluminal vesicles (ILVs), a favorable environment for endosomal escape. The identification of ILVs as a portal for passing proteins into the cytosol will aid the development of next-generation biologics that overcome the limitations imposed by cellular membranes. RESULTS Evaluating endosomal damageThe simplest way for a CPMP to escape from an endosome is if the endosome ruptures, in part or in full (25). Although there has been limited work on the effects of certain CPPs on the integrity of large unilamellar vesicles (LUVs) in vitro (26), the concentration-dependent effects of CPMPs or more traditional cell-penetrating peptides (CPPs) on endosomal integrity in cultured cells have not been thoroughly evaluated. Thus, we began our analysis with two complementary assays that together detect both subtle and severe endosomal damage in cells treated with a CPMP or CPP. One assay exploits a set of eGFP-labeled galectins to fluorescently tag damaged endosomes to enable their visualization using confocal microscopy, while the other employs a fluorescently tagged version of the nonalysine (Lys9) peptide to quantify the extent of endosome rupture in cells treated with a CPMP or CPP. In both cases, the effects of the two most efficient ...

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The vacuolar protein sorting genes in insects: A comparative genome view

Cited by 28 publications

References 119 publications

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

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