STAT3 in acute myeloid leukemia facilitates natural killer cell-mediated surveillance.

Acute myeloid leukemia (AML) is a heterogenous disease characterized by the clonal expansion of myeloid progenitor cells. Despite recent advancements in the treatment of AML, relapse still remains a significant challenge, necessitating the development of innovative therapies to eliminate minimal residual disease. One promising approach to address these unmet clinical needs is natural killer (NK) cell immunotherapy. To implement such treatments effectively, it is vital to comprehend how AML cells escape the NK-cell surveillance. Signal transducer and activator of transcription 3 (STAT3), a component of the Janus kinase (JAK)-STAT signaling pathway, is well-known for its role in driving immune evasion in various cancer types. Nevertheless, the specific function of STAT3 in AML cell escape from NK cells has not been deeply investigated. In this study, we unravel a novel role of STAT3 in sensitizing AML cells to NK-cell surveillance. We demonstrate that STAT3-deficient AML cell lines are inefficiently eliminated by NK cells. Mechanistically, AML cells lacking STAT3 fail to form an immune synapse as efficiently as their wild-type counterparts due to significantly reduced surface expression of intercellular adhesion molecule 1 (ICAM-1). The impaired killing of STAT3-deficient cells can be rescued by ICAM-1 overexpression proving its central role in the observed phenotype. Importantly, analysis of our AML patient cohort revealed a positive correlation between ICAM1 and STAT3 expression suggesting a predominant role of STAT3 in ICAM-1 regulation in this disease. In line, high ICAM1 expression correlates with better survival of AML patients underscoring the translational relevance of our findings. Taken together, our data unveil a novel role of STAT3 in preventing AML cells from escaping NK-cell surveillance and highlight the STAT3/ICAM-1 axis as a potential biomarker for NK-cell therapies in AML.

Pathophysiological Roles of Auxiliary Calcium Channel α2δ Subunits.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels, which are essential components of excitable cells such as skeletal and heart muscles, nerve cells of the brain and the peripheral nervous system, as well as endocrine cells. Over the recent years, α2δ proteins have been identified as critical regulators of synaptic functions, including the formation and differentiation of synapses. These functions require signalling mechanisms which are partly independent of calcium channels. Hence, in light of these features it is not surprising that the genes encoding for the four α2δ isoforms have recently been linked to neurological and neurodevelopmental disorders including epilepsy, autism spectrum disorders, schizophrenia, and depressive and bipolar disorders. Despite the increasing number of identified disease-associated mutations, the underlying pathophysiological mechanisms are only beginning to emerge. However, a thorough understanding of the pathophysiological role of α2δ proteins ideally serves two purposes: first, it will contribute to our understanding of general pathological mechanisms in synaptic disorders. Second, it may support the future development of novel and specific treatments for brain disorders. In this context, it is noteworthy that the antiepileptic and anti-allodynic drugs gabapentin and pregabalin both act via binding to α2δ proteins and are among the top sold drugs for treating neuropathic pain. In this book chapter, we will discuss recent developments in our understanding of the functions of α2δ proteins, both as calcium channel subunits and as independent regulatory entities. Furthermore, we present and summarize recently identified and likely pathogenic mutations in the genes encoding α2δ proteins and discuss potential underlying pathophysiological consequences at the molecular and structural level.

Structural insights into crista junction formation by the Mic60-Mic19 complex.

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie-shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function.

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites.

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.

The molecular mechanisms mediating class II PI 3-kinase function in cell physiology.

The phosphoinositide 3-kinase (PI3K) family of lipid-modifying enzymes plays vital roles in cell signaling and membrane trafficking through the production of 3-phosphorylated phosphoinositides. Numerous studies have analyzed the structure and function of class I and class III PI3Ks. In contrast, we know comparably little about the structure and physiological functions of the class II enzymes. Only recent studies have begun to unravel their roles in development, endocytic and endolysosomal membrane dynamics, signal transduction, and cell migration, while the mechanisms that control their localization and enzymatic activity remain largely unknown. Here, we summarize our current knowledge of the class II PI3Ks and outline open questions related to their structure, enzymatic activity, and their physiological and pathophysiological functions.

Structure and assembly of the mitochondrial membrane remodelling GTPase Mgm1.

Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.

Regulated membrane remodeling by Mic60 controls formation of mitochondrial crista junctions.

The mitochondrial contact site and cristae organizing system (MICOS) is crucial for the formation of crista junctions and mitochondrial inner membrane architecture. MICOS contains two core components. Mic10 shows membrane-bending activity, whereas Mic60 (mitofilin) forms contact sites between inner and outer membranes. Here we report that Mic60 deforms liposomes into thin membrane tubules and thus displays membrane-shaping activity. We identify a membrane-binding site in the soluble intermembrane space-exposed part of Mic60. This membrane-binding site is formed by a predicted amphipathic helix between the conserved coiled-coil and mitofilin domains. The mitofilin domain negatively regulates the membrane-shaping activity of Mic60. Binding of Mic19 to the mitofilin domain modulates this activity. Membrane binding and shaping by the conserved Mic60-Mic19 complex is crucial for crista junction formation, mitochondrial membrane architecture and efficient respiratory activity. Mic60 thus plays a dual role by shaping inner membrane crista junctions and forming contact sites with the outer membrane.

Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions.

The cytosolic tripartite NLR receptors serve as important signalling platforms in innate immunity. While the C-terminal domains act as sensor and activation modules, the N-terminal death-like domain, e.g. the CARD or pyrin domain, is thought to recruit downstream effector molecules by homotypic interactions. Such homotypic complexes have been determined for all members of the death-domain superfamily except for pyrin domains. Here, crystal structures of human NLRP14 pyrin-domain variants are reported. The wild-type protein as well as the clinical D86V mutant reveal an unexpected rearrangement of the C-terminal helix α6, resulting in an extended α5/6 stem-helix. This reordering mediates a novel symmetric pyrin-domain dimerization mode. The conformational switching is controlled by a charge-relay system with a drastic impact on protein stability. How the identified charge relay allows classification of NLRP receptors with respect to distinct recruitment mechanisms is discussed.

Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code.

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.

Functional mapping of human dynamin-1-like GTPase domain based on x-ray structure analyses.

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.

Structural and functional analysis of the NLRP4 pyrin domain.

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2-α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD-PYD interaction specificity.