Direct Activation of Human MLKL by a Select Repertoire of Inositol Phosphate Metabolites

McNamara, Dan E.; Dovey, Cole M.; Hale, Andrew T.; Quarato, Giovanni; Grace, Christy R.; Guibao, Cristina D.; Diep, Jonathan; Nourse, Amanda; Cai, Casey R; Wu, Hen-Ming; Kalathur, Ravi C.; Green, Douglas R.; York, John D.; Carette, Jan E.; Moldoveanu, Tudor

doi:10.1016/j.chembiol.2019.03.010

Cited by 45 publications

(86 citation statements)

References 101 publications

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“…4). These residues reside on the α4 helix of the MLKL 4HB domain, on the face opposite from the two clusters of basic residues previously implicated in phospholipid and inositol phosphate binding (14,(30)(31)(32)(33), indicating that they perform distinct functions from mediating membrane association and activation by inositol phosphates. Previous alanine scanning mutagenesis of the mouse MLKL NTR to introduce R105A/D106A, E109A/E110A, and LLLL112-115AAAA (SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 94%

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

Petrie

Birkinshaw

Koide

et al. 2020

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

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The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

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Section: Discussionmentioning

confidence: 94%

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

Petrie

Birkinshaw

Koide

et al. 2020

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

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“…IP6 is found in mammalian cells at concentrations of 10-100uM [11], and is synthesized by a series of host enzymes through a complex and not fully resolved process (Fig 1A) [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. The immediate precursor to IP6 is inositol pentakisphosphate (I(1,3,4,5,6)P 5 or IP5), and the only enzyme known to catalyze the addition of the final 2-phosphate is inositol-pentakisphosphate 2-kinase (IPPK) [12][13][14][15][16].…”

Section: Plos Pathogensmentioning

confidence: 99%

“…Recently, a genetic screen performed to identify genes involved in necroptosis revealed that inositol phosphates IP5 or IP6 are required for this process [21]. Importantly, the screen identified the genes IPMK and inositol-tetrakisphosphate 1-kinase (ITPK1) as being required for necroptosis, and cells lacking either of these genes were noticeably deficient in IP5 and IP6 [21,22]. Thus, IPMK and ITPK1 likely cooperate in the production of IP5 in cells.…”

Section: Plos Pathogensmentioning

confidence: 99%

Primate lentiviruses require Inositol hexakisphosphate (IP6) or inositol pentakisphosphate (IP5) for the production of viral particles

et al. 2020

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Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.

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“…In addition, inositol phosphates (IPs), which are soluble intermediates of lipid metabolism, also modulate MLKL necroptotic activity ( Figure 2). Specifically, the highly phosphorylated versions (i.e., IP6 > IP5 > IP4) can bind to MLKL, thereby promoting a conformational change that exposes the killer (4HB) domain by releasing the auto-inhibition by the adjacent brace regions [39,40]. In addition, it has been found that very-long-chain fatty acids promote necroptosis, probably due to their effect on membrane organization that would potentially modulate MLKL activity [15].…”

Section: Membrane Permeabilization By Mlkl In Necroptosismentioning

confidence: 99%

Partners in Crime: The Interplay of Proteins and Membranes in Regulated Necrosis

Ros

Pedrera

García‐Sáez

2020

IJMS

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Pyroptosis, necroptosis, and ferroptosis are well-characterized forms of regulated necrosis that have been associated with human diseases. During regulated necrosis, plasma membrane damage facilitates the movement of ions and molecules across the bilayer, which finally leads to cell lysis and release of intracellular content. Therefore, these types of cell death have an inflammatory phenotype. Each type of regulated necrosis is mediated by a defined machinery comprising protein and lipid molecules. Here, we discuss how the interaction and reshaping of these cellular components are essential and distinctive processes during pyroptosis, necroptosis, and ferroptosis. We point out that although the plasma membrane is the common target in regulated necrosis, different mechanisms of permeabilization have emerged depending on the cell death form. Pore formation by gasdermins (GSDMs) is a hallmark of pyroptosis, while mixed lineage kinase domain-like (MLKL) protein facilitates membrane permeabilization in necroptosis, and phospholipid peroxidation leads to membrane damage in ferroptosis. This diverse repertoire of mechanisms leading to membrane permeabilization contributes to define the specific inflammatory and immunological outcome of each type of regulated necrosis. Current efforts are focused on new therapies that target critical protein and lipid molecules on these pathways to fight human pathologies associated with inflammation.

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Direct Activation of Human MLKL by a Select Repertoire of Inositol Phosphate Metabolites

Cited by 45 publications

References 101 publications

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

Primate lentiviruses require Inositol hexakisphosphate (IP6) or inositol pentakisphosphate (IP5) for the production of viral particles

Partners in Crime: The Interplay of Proteins and Membranes in Regulated Necrosis

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