Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.
The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.
The
life cycle of the human immunodeficiency virus type 1 (HIV-1)
has an absolute requirement for ribosomal frameshifting during protein
translation in order to produce the polyprotein precursor of the viral
enzymes. While an RNA stem-loop structure (the “HIV-1 Frameshift
Stimulating Signal”, or HIV-1 FSS) controls the frameshift
efficiency and has been hypothesized as an attractive therapeutic
target, developing compounds that selectively bind this RNA and interfere
with HIV-1 replication has proven challenging. Building on our prior
discovery of a “hit” molecule able to bind this stem-loop,
we now report the development of compounds displaying high affinity
for the HIV-1 FSS. These compounds are able to enhance frameshifting
more than 50% in a dual-luciferase assay in human embryonic kidney
cells, and they strongly inhibit the infectivity of pseudotyped HIV-1
virions.
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