The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of ...autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1’s function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1’s functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1’s transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.
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•Autoinhibited (3.3 Å) and active (6.8 Å) structures of pro-degenerative NADase SARM1 solved•Identification of a critical autoinhibitory lock•Lock mutations convert inactive SARM1 to an active, neurotoxic state•Enzymatic studies explain SARM1’s functional dependence on local metabolic environment
Bratkowski et al. describe cryo-EM structures of autoinhibited and active SARM1, an axon-enriched, injury-activated NADase. Structure-function studies elucidate the mode of autoinhibition and SARM1 activity regulation. The authors provide mechanistic insight into SARM1’s regulation by the local metabolic environment, laying the foundation for future SARM1-based therapies to treat neurological diseases.
There is currently no reliable and easily applicable diagnostic marker for Parkinson's disease (PD). The aims of the present study were to compare the expression profiles of the microRNA29 family ...(miR-29s) in blood serum from patients with PD with healthy controls and to clarify whether the expression of miR-29s is correlated with disease severity, duration or L-dopa therapy and whether expression depends on the gender and age of patients. The levels of blood serum miR-29s in 80 patients with PD and 80 unaffected controls were assessed by reverse transcription-quantitative real-time PCR. The PCR products were confirmed by cloning and sequencing. Additionally, the expression of miR-7 in the blood serum from PD patients and control subjects was assessed. Serum miR-29 levels were significantly downregulated in PD patients compared to healthy controls. The serum miR-29 levels in female PD patients were markedly higher than in male PD patients. The expression of serum miR-29a and miR-29c expression tended to decrease with disease severity. Moreover, we found that serum miR-7 levels did not differ between PD patients and control subjects. Therefore, the reduction of serum miR-29 levels, particularly miR-29a and miR-29c, warrants further investigation of its potential serving as biomarkers for PD.
A structural model of a Ras-Raf signalosome Mysore, Venkatesh P; Zhou, Zhi-Wei; Ambrogio, Chiara ...
Nature structural & molecular biology,
10/2021, Letnik:
28, Številka:
10
Journal Article
Recenzirano
Odprti dostop
The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, ...multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.
The OX
orexin receptor (OX
R) is a highly expressed G protein-coupled receptor (GPCR) in the brain that regulates wakefulness and circadian rhythms in humans. Antagonism of OX
R is a proven ...therapeutic strategy for insomnia drugs, and agonism of OX
R is a potentially powerful approach for narcolepsy type 1, which is characterized by the death of orexinergic neurons. Until recently, agonism of OX
R had been considered 'undruggable.' We harness cryo-electron microscopy of OX
R-G protein complexes to determine how the first clinically tested OX
R agonist TAK-925 can activate OX
R in a highly selective manner. Two structures of TAK-925-bound OX
R with either a G
mimetic or G
reveal that TAK-925 binds at the same site occupied by antagonists, yet interacts with the transmembrane helices to trigger activating microswitches. Our structural and mutagenesis data show that TAK-925's selectivity is mediated by subtle differences between OX
and OX
receptor subtypes at the orthosteric pocket. Finally, differences in the polarity of interactions at the G protein binding interfaces help to rationalize OX
R's coupling selectivity for G
signaling. The mechanisms of TAK-925's binding, activation, and selectivity presented herein will aid in understanding the efficacy of small molecule OX
R agonists for narcolepsy and other circadian disorders.
The characteristic brain pathology and motor and nonmotor symptoms of Parkinson’s disease (PD) are well established. However, the details regarding the causes of the disease and its course are much ...less clear. Animal models have significantly enriched our current understanding of the progression of this disease. Among various neurotoxin-based models of PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model is the most commonly studied model. Here, we provide an overview of the dynamic changes in the nigrostriatal pathway in the MPTP mouse model of PD. Pathophysiological events, such as reductions in the striatal dopamine (DA) concentrations and levels of the tyrosine hydroxylase (TH) protein, depletion of TH-positive nerve fibers, a decrease in the number of TH-positive neurons in the substantia nigra pars compacta (SNpc), and glial activation, are addressed. This article will assist with the development of interventions or therapeutic strategies for PD.
Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors that have been shown not only to play a key role in regulating normal cellular processes, but also to be critical in the ...development and progression of many types of human cancer. To date, many crystal structures of individual domains of RTKs are available; however, the high resolution structural information for full‐length RTK proteins either in their apo‐state or in ligand‐bound states remains elusive. In addition, there are no structural detail about how the active full‐length receptor can bind the effector protein for the activation of downstream signaling pathway. Lacking a complete structure of full‐length receptor signaling complex hinder our understanding of how this important protein functions. We determine the structures of full‐length RTK proteins, in complex with their ligands and downstream proteins, by single particle cryo‐EM, as key to understand the critical role that this protein family plays in the physiology of the cell. Epidermal growth factor receptor (EGFR), RET, cMET and Insulin receptor (IR), the most studied members in the RTK family, are chosen as the targets for structure determination. These pathways comprise a complex and diverse signaling network, and play an essential role in normal cell proliferation, differentiation, development and motility. Aberrant activation can lead to both tumor growth and metastatic progression of cancer cells, which makes them promising candidate for cancer therapy. Determination of full‐length RTKs in apo‐ and ligand‐bound states shed light on its mechanisms of kinase activation, enhance understanding of the interaction between receptor and their ligand and inhibitors, and facilitate development of selective anti‐cancer drugs. Reconstitution of fully assembled signaling complex reveal the mechanism by which the active receptors initialize the downstream signaling transudation, and provide direct structural evidence for how the plasma membrane plays roles in this processing. Solving the structures of different members of the RTK family reveal similarities and differences in the structure‐function relationships within this receptor family, which can potentially lead to new strategies to selectively inhibit/modulate individual pathways for cancer therapy or concurrently target various pathways, an approach that has been suggested to improve therapeutic effectiveness.
Support or Funding Information
the Welch foundation, and CPRIT foundation
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive degeneration of midbrain dopaminergic neurons. The miR-29s family, including
miR-29a
and
miR-29b1
as well as
...miR-29b2
and
miR-29c
, are implicated in aging, metabolism, neuronal survival, and neurological disorders. In this study, the roles of
miR-29a/b1
in aging and PD were investigated.
miR-29a/b1
knockout mice (named as 29a KO hereafter) and their wild-type (WT) controls were used to analyze aging-related phenotypes. After challenged with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), dopaminergic injuries, glial activation, and mouse behaviors were evaluated. Primary glial cells were further cultured to explore the underlying mechanisms. Additionally, the levels of miR-29s in the cerebrospinal fluid (CSF) of PD patients (
n
= 18) and healthy subjects (
n
= 17) were quantified. 29a KO mice showed dramatic weight loss, kyphosis, and along with increased and deepened wrinkles in skins, when compared with WT mice. Moreover, both abdominal and brown adipose tissues reduced in 29a KO mice, compared to their WT counterpart. However, in MPTP-induced PD mouse model, the deficiency of
miR-29a/b1
led to less severe damages of dopaminergic system and mitigated glial activation in the nigrostriatal pathway, and subsequently alleviated the motor impairments in 3-month-old mice. Eight-month-old mutant mice maintained such a resistance to MPTP intoxication. Mechanistically, the deficiency of
miR-29a/b-1
promoted the expression of neurotrophic factors in 1-Methyl-4-phenylpyridinium (MPP
+
)-treated primary mixed glia and primary astrocytes. In lipopolysaccharide (LPS)-treated primary microglia, knockout of
miR-29a/b-1
inhibited the expression of inflammatory factors, and promoted the expression of anti-inflammatory factors and neurotrophic factors. Knockout of
miR-29a/b1
increased the activity of AMP-activated protein kinase (AMPK) and repressed NF-κB/p65 signaling in glial cells. Moreover, we found miR-29a level was increased in the CSF of patients with PD. Our results suggest that 29a KO mice display the peripheral premature senility. The combined effects of less activated glial cells might contribute to the mitigated inflammatory responses and elicit resistance to MPTP intoxication in
miR-29a/b1
KO mice.
The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy ...(cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.
ObjectivePrevious studies have shown that ex utero intrapartum therapy (EXIT) is safe and feasible for newborns with congenital diaphragmatic hernia (CDH). This study reports our experience with EXIT ...in fetuses with CDH in an attempt to explore the efficacy of EXIT on the survival rate of this population.MethodsA retrospective analysis of the clinical data of 116 children with CDH was conducted. The children were assigned to EXIT and non-EXIT groups. Propensity score matching (PSM) toward clinical data was performed, and the clinical characteristics and outcomes were compared. Taking survival at discharge as the main outcome, logistic regression analysis was carried out to explore the efficacy of EXIT on survival.ResultsDuring the study period, 30 of 116 children received EXIT. After PSM, the survival rates of the EXIT group and the non-EXIT group were 82.76% (24/29) and 48.28% (14/29), respectively (p=0.006). EXIT (OR=0.083, 95% CI=0.013to 0.525, p=0.008), liver herniation (OR=16.955, 95% CI=2.342 to 122.767, p=0.005), and gestational age at diagnosis (OR=0.662, 95% CI=0.497 to 0.881, p=0.005) were independent mortality-related risk factors of all children with CDH. Ninety-nine of 116 children underwent surgery. After PSM, the postoperative survival rates of the EXIT group and non-EXIT group were 84.6% (22/26) and 76.9% (20/26), respectively (p=0.754). Liver herniation (OR=10.451, 95% CI=1.641 to 66.544, p=0.013) and gestational age at diagnosis (OR=0.736, 95% CI=0.577 to 0.938, p=0.013) were independent mortality-related risk factors of children after surgery.ConclusionEXIT can be performed safely for selected prenatally diagnosed CDH neonates with potentially better survival and does not cause more maternal complications compared with traditional cesarean section.
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