Human Type 3 Iodothyronine Selenodeiodinase Is Located in the Plasma Membrane and Undergoes Rapid Internalization to Endosomes

Abstract Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4–rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4–rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.

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A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates

Clinical and Vaccine Immunology ◽

10.1128/cvi.00150-16 ◽

2016 ◽

Vol 23 (7) ◽

pp. 586-600 ◽

Cited By ~ 10

Author(s):

Jian Sha ◽

Michelle L. Kirtley ◽

Curtis Klages ◽

Tatiana E. Erova ◽

Maxim Telepnev ◽

...

Keyword(s):

Fusion Gene ◽

The United States ◽

Pneumonic Plague ◽

Content Type ◽

Human Type ◽

Protective Antigens ◽

Type 3 Secretion System ◽

Needle Protein ◽

Trivalent Vaccine ◽

Type 3

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens ofYersinia pestis. We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimizedlcrVor the fusion gene designatedYFV(consisting ofycsF,caf1, andlcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged withY. pestisCO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

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Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection

mBio ◽

10.1128/mbio.00877-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 5

Author(s):

Brian C. Russo ◽

Jeffrey K. Duncan ◽

Marcia B. Goldberg

Keyword(s):

Plasma Membrane ◽

Protein Secretion ◽

Secretion System ◽

Bacterial Virulence ◽

Eukaryotic Cells ◽

Effector Protein ◽

Virulence Proteins ◽

Type 3 Secretion System ◽

Type 3 ◽

Pore Protein

ABSTRACTMany Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of theShigellatranslocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes.Salmonella entericaserovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to theSalmonellatranslocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection.IMPORTANCEThe type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of theShigellatranslocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.

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Cryo-EM structure of human type-3 inositol triphosphate receptor reveals the presence of a self-binding peptide that acts as an antagonist

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011570 ◽

2020 ◽

Vol 295 (6) ◽

pp. 1743-1753 ◽

Cited By ~ 5

Author(s):

Caleigh M. Azumaya ◽

Emily A. Linton ◽

Caitlin J. Risener ◽

Terunaga Nakagawa ◽

Erkan Karakas

Keyword(s):

Calcium Signaling ◽

Metabolic Diseases ◽

Inositol Triphosphate ◽

Biological Processes ◽

Human Type ◽

Physiological Processes ◽

Molecular Determinant ◽

Specific Regulation ◽

Type 3

Calcium-mediated signaling through inositol 1,4,5-triphosphate receptors (IP3Rs) is essential for the regulation of numerous physiological processes, including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Deregulation of IP3Rs leads to pathological calcium signaling and is implicated in many common diseases, including cancer and neurodegenerative, autoimmune, and metabolic diseases. Revealing the mechanism of activation and inhibition of this ion channel will be critical to an improved understanding of the biological processes that are controlled by IP3Rs. Here, we report structural findings of the human type-3 IP3R (IP3R-3) obtained by cryo-EM (at an overall resolution of 3.8 Å), revealing an unanticipated regulatory mechanism where a loop distantly located in the primary sequence occupies the IP3-binding site and competitively inhibits IP3 binding. We propose that this inhibitory mechanism must differ qualitatively among IP3R subtypes because of their diverse loop sequences, potentially serving as a key molecular determinant of subtype-specific calcium signaling in IP3Rs. In summary, our structural characterization of human IP3R-3 provides critical insights into the mechanistic function of IP3Rs and into subtype-specific regulation of these important calcium-regulatory channels.

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Plasma and intracellular membrane inositol 1,4,5-trisphosphate receptors mediate the Ca2+ increase associated with the ATP-induced increase in ciliary beat frequency

AJP Cell Physiology ◽

10.1152/ajpcell.00343.2003 ◽

2004 ◽

Vol 287 (4) ◽

pp. C1114-C1124 ◽

Cited By ~ 34

Author(s):

Nelson P. Barrera ◽

Bernardo Morales ◽

Manuel Villalón

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Protein Kinase ◽

Beat Frequency ◽

Ciliary Beat Frequency ◽

Receptor Type ◽

Ciliated Cells ◽

Inositol 1,4,5 Trisphosphate ◽

Type 3 ◽

Ciliary Beat

An increase in intracellular free Ca2+ concentration ([Ca2+]i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP3) receptor-dependent Ca2+ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP3 peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca2+ from internal stores and an influx of extracellular Ca2+, respectively. In addition, ATP-dependent Ca2+ mobilization required protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP3 receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca2+]i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca2+ influx is also dependent on IP3 receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP3 receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP3 receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP3 receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP3 receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP3 receptor can directly trigger Ca2+ influx in response to ATP.

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Evaluation of the human type 3 adenoviral dodecahedron as a vector to target acute myeloid leukemia

Molecular Therapy — Methods & Clinical Development ◽

10.1016/j.omtm.2020.11.009 ◽

2021 ◽

Vol 20 ◽

pp. 181-190

Author(s):

Benjamin Caulier ◽

Gaëlle Stofleth ◽

Dalil Hannani ◽

Mélanie Guidetti ◽

Véronique Josserand ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Human Type ◽

Type 3 ◽

Acute Myeloid

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Human Types 1 and 3 3α-Hydroxysteroid Dehydrogenases: Differential Lability and Tissue Distribution1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.86.2.7216 ◽

2001 ◽

Vol 86 (2) ◽

pp. 841-846 ◽

Cited By ~ 1

Author(s):

Isabelle Dufort ◽

Fernand Labrie ◽

Van Luu-The

Keyword(s):

Hacat Keratinocytes ◽

Human Embryonic Kidney ◽

Intact Cells ◽

Hek 293 ◽

Human Type ◽

Hydroxysteroid Dehydrogenases ◽

293 Cells ◽

Hydroxy Compounds ◽

Type 3

3α-Hydroxysteroid dehydrogenases (3α-HSDs) catalyze the conversion of 3-ketosteroids to 3α-hydroxy compounds. The best known 3α-HSD activity is the transformation of the most potent natural androgen, dihydrotestosterone, into 5α-androstan-3α,17β-diol (3α-diol), a compound having much lower activity. Previous reports show that 3α-HSDs are involved in the metabolism of glucocorticoids, progestins, prostaglandins, bile acid precursors, and xenobiotics. 3α-HSDs could, thus, play a crucial role in the control of a series of active steroid levels in target tissues. In the human, type 1 3α-HSD was first identified as human chlordecone reductase. Recently, we have isolated and characterized type 3 3α-HSD that shares 81.7% identity with human type 1 3α-HSD. The transfection of vectors expressing types 1 and 3 3α-HSD in transformed human embryonic kidney (HEK-293) cells indicates that both enzymes efficiently catalyze the transformation of dihydrotestosterone into 3α-diol in intact cells. However, when the cells are broken, the activity of type 3 3α-HSD is rapidly lost, whereas the type 1 3α-HSD activity remains stable. We have previously found that human type 5 17β-HSD which possesses 84% and 86% identity with types 1 and 3 3α-HSD, respectively, is also labile, whereas rodent enzymes such as mouse type 5 17β-HSD and rat 3α-HSD are stable after homogenization of the cells. The variable stability of different enzymatic activities in broken cell preparations renders the comparison of different enzymes difficult. RNA expression analysis indicates that human type 1 3α-HSD is expressed exclusively in the liver, whereas type 3 is more widely expressed and is found in the liver, adrenal, testis, brain, prostate, and HaCaT keratinocytes. Based on enzymatic characteristics and sequence homology, it is suggested that type 1 3α-HSD is an ortholog of rat 3α-HSD while type 3 3α-HSD, which must have diverged recently, seems unique to human and is probably more involved in intracrine activity.

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