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

2018 ◽  
Author(s):  
Angela Steinauer ◽  
Jonathan R. LaRochelle ◽  
Rebecca Wissner ◽  
Samuel Berry ◽  
Alanna Schepartz
Keyword(s):  
Cell Penetrating Peptides ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Endosomal Escape ◽  
Stapled Peptides ◽  
Critical Determinant ◽  
Small Proteins ◽  
Primary Means ◽  
The Difference

AbstractProtein 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.

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

2019 ◽  
Vol 116 (2) ◽  
pp. 512-521 ◽  
Author(s):  
Angela Steinauer ◽  
Jonathan R. LaRochelle ◽  
Susan L. Knox ◽  
Rebecca F. Wissner ◽  
Samuel Berry ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Endosomal Escape ◽  
Stapled Peptides ◽  
Critical Determinant ◽  
Small Proteins ◽  
Primary Means

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 cell-based 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.

scholarly journals Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway

2020 ◽  
Vol 15 (9) ◽  
pp. 2355-2363 ◽  
Author(s):  
Helena M. Kondow-McConaghy ◽  
Nandhini Muthukrishnan ◽  
Alfredo Erazo-Oliveras ◽  
Kristina Najjar ◽  
Rudolph L. Juliano ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Endocytic Pathway ◽  
Endosomal Escape ◽  
Cell Penetrating

scholarly journals Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

2021 ◽  
Vol 14 (8) ◽  
pp. 757
Author(s):  
Iga Jakobowska ◽  
Frank Becker ◽  
Stefano Minguzzi ◽  
Kerrin Hansen ◽  
Björn Henke ◽  
...  
Keyword(s):  
Rate Constants ◽  
Cross Correlation ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Binding Kinetics ◽  
Acceptor Site ◽  
Lactate Transport ◽  
Entry Site ◽  
Hydrogen Bond Acceptor

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

scholarly journals Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

2017 ◽  
Vol 8 (3-4) ◽  
pp. 131-141 ◽  
Author(s):  
Julia C. LeCher ◽  
Scott J. Nowak ◽  
Jonathan L. McMurry
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Great Promise ◽  
Delivery Methods ◽  
Oligomeric Proteins ◽  
Cell Penetrating ◽  
History Of ◽  
A Cell ◽  
Escape Problem ◽  
Primary Focus

AbstractCell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This ‘endosomal escape problem’ has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

scholarly journals Batteries in a vehicle – conditions, capabilities and limitations

2018 ◽  
Vol 180 ◽  
pp. 03001
Author(s):  
Piotr Julian Biczel ◽  
Maciej Kwiatkowski
Keyword(s):  
Specific Energy ◽  
Electric Motor ◽  
Combustion Engine ◽  
Mass Range ◽  
Electrochemical Cells ◽  
Charging Time ◽  
Vehicle Mass ◽  
Primary Means ◽  
The Difference ◽  
Basic Features

Electric buses are now seen as the primary means of public transport in cities. However, their exploitation is associated with a number of limitations. The article presents the problem of battery use in buses. The design differences between the vehicle with the internal combustion engine and the electric motor are discussed. In particular, authors compared the number of passengers that both types of buses can take, as well as the difference in vehicle mass and range, and the reasons for these differences. The types of electrochemical cells that can be used to power vehicles and their basic features are presented. The article focuses on the NMC, LFP, LTO and LIC type cells. Next, the battery pack structure and its components were described. The specific energies of cells and batteries constructed from these cells were compared. Next, the most important features of the vehicle equipped with batteries with various types of cells are discussed. The mass, range, charging time and cycle lifetime of the batteries were compared. The basic dependence was noted: the greater the specific energy, the smaller the number of battery cycles. So if the bus has to take more passengers, it must have light batteries, which should be frequently charged, which requires high cyclic lifetimes.

Decoding the Entry of Two Novel Cell-Penetrating Peptides in HeLa Cells:  Lipid Raft-Mediated Endocytosis and Endosomal Escape†

Biochemistry ◽  
2005 ◽  
Vol 44 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Christina Foerg ◽  
Urs Ziegler ◽  
Jimena Fernandez-Carneado ◽  
Ernest Giralt ◽  
Robert Rennert ◽  
...  
Keyword(s):  
Lipid Raft ◽  
Hela Cells ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Cell Penetrating

scholarly journals Aggregatibacter actinomycetemcomitansLtxA hijacks endocytic trafficking pathways in human lymphocytes

2019 ◽  
Author(s):  
Edward T Lally ◽  
Kathleen Boesze-Battaglia ◽  
Anuradha Dhingra ◽  
Nestor M Gomez ◽  
Claire H Mitchell ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Human Lymphocytes ◽  
Membrane Damage ◽  
Confocal Imaging ◽  
Endocytic Pathway ◽  
Jurkat Cells ◽  
Endocytic Trafficking ◽  
Recycling Endosomes ◽  
Plasma Membrane Damage ◽  
Human T Lymphocyte

ABSTRACTLeukotoxin (LtxA) from oral pathogenAggregatibacter actinomycetemcomitansis a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18) expressing leukocytes and ultimately causes cell death; however toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5 knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pH. Our results demonstrate that LtxA/LFA-1 complex gains an access to the cytosol of Jurkat cells without evidence of plasma membrane damage utilizing dynamin-dependent and clathrin-independent mechanism. Upon internalization LtxA follows the LFA-1 endocytic trafficking pathways as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp2) and CD11a. Knockdown of Rab5a resulted in loss of susceptibility of Jurkat cells to LtxA cytotoxicity suggesting that late events of LtxA endocytic trafficking are required for toxicity. The toxin trafficking via the degradation endocytic pathway may culminate in delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.

scholarly journals The mobility gap: estimating mobility levels required to control Canada’s winter COVID-19 surge

2021 ◽  
Author(s):  
Kevin A. Brown ◽  
Jean-Paul R. Soucy ◽  
Sarah A. Buchan ◽  
Shelby L. Sturrock ◽  
Isha Berry ◽  
...  
Keyword(s):  
Growth Rate ◽  
Residential Mobility ◽  
Rate Ratio ◽  
Herd Immunity ◽  
Primary Means ◽  
Low Levels ◽  
The Difference ◽  
Canadian Provinces ◽  
Pharmaceutical Interventions ◽  
Mobility Gap

AbstractBackgroundNon-pharmaceutical interventions remain a primary means of suppressing COVID-19 until vaccination coverage is sufficient to achieve herd immunity. We used anonymized smartphone mobility measures in seven Canadian provinces to quantify the mobility level needed to suppress COVID-19 (mobility threshold), and the difference relative to current mobility levels (mobility gap).MethodsWe conducted a longitudinal study of weekly COVID-19 incidence from March 15, 2020 to January 16, 2021, among provinces with 20 COVID-19 cases in at least 10 weeks. The outcome was weekly growth rate defined as the ratio of current cases compared to the previous week. We examined the effects of average time spent outside the home (non-residential mobility) in the prior three weeks using a lognormal regression model accounting for province, season, and mean temperature. We calculated the COVID-19 mobility threshold and gap.ResultsAcross the 44-week study period, a total of 704,294 persons were infected with COVID-19. Non-residential mobility dropped rapidly in the spring and reached a median of 36% (IQR: 31,40) in April 2020. After adjustment, each 5% increase in non-residential mobility was associated with a 9% increase in the COVID-19 weekly growth rate (ratio=1.09, 95%CI: 1.07,1.12). The mobility gap increased through the fall months, which was associated with increasing case growth.InterpretationMobility strongly and consistently predicts weekly case growth, and low levels of mobility are needed to control COVID-19 through winter 2021. Mobility measures from anonymized smartphone data can be used to guide the provincial and regional implementation and loosening of physical distancing measures.

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