Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of ...inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance. Importance: RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.
An expanded CAG repeat is the underlying genetic defect in Huntington disease, a disorder characterized by motor, psychiatric and cognitive deficits and striatal atrophy associated with neuronal ...loss. An accurate animal model of this disease is crucial for elucidation of the underlying natural history of the illness and also for testing experimental therapeutics. We established a new yeast artificial chromosome (YAC) mouse model of HD with the entire human HD gene containing 128 CAG repeats (YAC128) which develops motor abnormalities and age-dependent brain atrophy including cortical and striatal atrophy associated with striatal neuronal loss. YAC128 mice exhibit initial hyperactivity, followed by the onset of a motor deficit and finally hypokinesis. The motor deficit in the YAC128 mice is highly correlated with striatal neuronal loss, providing a structural correlate for the behavioral changes. The natural history of HD-related changes in the YAC128 mice has been defined, demonstrating the presence of huntingtin inclusions after the onset of behavior and neuropathological changes. The HD-related phenotypes of the YAC128 mice show phenotypic uniformity with low inter-animal variability present, which together with the age-dependent striatal neurodegeneration make it an ideal mouse model for the assessment of neuroprotective and other therapeutic interventions.
Background: RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in the RGS2 gene in humans have been linked ...to hypertension, preeclampsia, and anxiety disorders. Mice deficient for Rgs2 (Rgs2 Null ) exhibit hypertension, anxiety, and altered adipose development and function. Methods: To study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for the Rgs2 gene ( Rgs2 Flox ) was developed. These mice were bred with mice expressing Cre-recombinase via the Agouti-related peptide locus ( Agrp -Cre) to cause deletion of Rgs2 from all cells expressing Agrp (Rgs2 Agrp-KO ), or a novel transgenic mouse expressing Cre-recombinase via the ANG (angiotensin) type 1A receptor ( Agtr1a / AT 1A ) promoter encoded in a bacterial artificial chromosome (BAC-AT 1A -Cre) to delete Rgs2 in all Agtr1a -expressing cells ( Rgs2 AT1A-KO ). Results: Whereas Rgs2 Flox , Rgs2 Agrp-KO , and BAC-AT 1A -Cre mice exhibited normal growth and survival, Rgs2 AT1A-KO exhibited pre-weaning lethality. Relative to littermates, Rgs2 Agrp-KO exhibited reduced fat gains when maintained on a high fat diet, associated with increased energy expenditure. Similarly, surviving adult Rgs2 AT1A-KO mice also exhibited increased energy expenditure. Surprisingly, given the hypertensive phenotype previously reported for Rgs2 Null mice and evidence supporting a role for RGS2 in terminating AT 1A signaling in various cell types, Rgs2 AT1A-KO mice exhibited normal blood pressure, ingestive behaviors, and renal functions, both before and after chronic infusion of ANG (490 ng/kg/min, sc). Conclusions: These results demonstrate the development of a novel mouse with conditional expression of Rgs2 and illustrate the role of Rgs2 within selected cell types for cardiometabolic control.
Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in ...either the mitochondrial or nuclear genome. Only 5 % of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (
C22orf16
) (later designated as
CHCHD10
) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in
cis
in
CHCHD10
which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that
CHCHD10
is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.
Genomic triplication of the α‐synuclein gene recently has been associated with familial Parkinson's disease in the Spellman–Muenter kindred. Here, we present an independent family, of ...Swedish‐American descent, with hereditary early‐onset parkinsonism with dementia due to α‐synuclein triplication. Brain tissue available from affected individuals in both kindreds provided the opportunity to compare their clinical, pathological, and biochemical phenotypes. Of note, studies of brain mRNA and soluble protein levels demonstrate a doubling of α‐synuclein expression, consistent with molecular genetic data. Pathologically, cornu ammonis 2/3 hippocampal neuronal loss appears to be a defining feature of this form of inherited parkinsonism. The profound implications of α‐synuclein overexpression for idiopathic synucleinopathies are discussed.
DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 ...(Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle-regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1-stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.
The type III secretion system (T3SS) is a macromolecular 'injectisome' that allows bacterial pathogens to transport virulence proteins into the eukaryotic host cell. This macromolecular complex is ...composed of connected ring-like structures that span both bacterial membranes. The crystal structures of the periplasmic domain of the outer membrane secretin EscC and the inner membrane protein PrgH reveal the conservation of a modular fold among the three proteins that form the outer membrane and inner membrane rings of the T3SS. This leads to the hypothesis that this conserved fold provides a common ring-building motif that allows for the assembly of the variably sized outer membrane and inner membrane rings characteristic of the T3SS. Using an integrated structural and experimental approach, we generated ring models for the periplasmic domain of EscC and placed them in the context of the assembled T3SS, providing evidence for direct interaction between the outer membrane and inner membrane ring components and an unprecedented span of the outer membrane secretin.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by many diverse genetic etiologies. Although therapeutics that specifically target causal mutations may rescue ...individual types of ALS, such approaches cannot treat most patients since they have unknown genetic etiology. Thus, there is a critical need for therapeutic strategies that rescue multiple forms of ALS. Here, we combine phenotypic chemical screening on a diverse cohort of ALS patient-derived neurons with bioinformatic analysis of large chemical and genetic perturbational datasets to identify broadly effective genetic targets for ALS. We show that suppressing the gene-encoding, spliceosome-associated factor SYF2 alleviates TDP-43 aggregation and mislocalization, improves TDP-43 activity, and rescues C9ORF72 and causes sporadic ALS neuron survival. Moreover, Syf2 suppression ameliorates neurodegeneration, neuromuscular junction loss, and motor dysfunction in TDP-43 mice. Thus, suppression of spliceosome-associated factors such as SYF2 may be a broadly effective therapeutic approach for ALS.
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•A phenotypic screen identifies SYF2 suppression as a therapeutic target for ALS•SYF2 suppression alleviates disease in a diverse collection of iPSC models of ALS•SYF2 suppression slows TDP-43 nuclear export, reduces aggregation, and improves splicing•Syf2 suppression mitigates motor deficits and neuropathology in a mouse model of ALS
Reducing levels of a spliceosome-associated factor, SYF2, attenuates TDP-43 pathology in models of diverse forms of ALS. Therefore, these findings by Linares et al. indicate that SYF2 suppression may function as a broadly acting therapeutic strategy for the treatment of ALS.
Mesenchymal stem/stromal cells (MSCs) play crucial roles in maintaining tissue homeostasis during physiological turnovers and injuries. Very little is known about the phenotype, distribution and ...molecular nature of MSCs in freshly isolated human salivary glands (SGs) as most reports have focused on the analysis of cultured MSCs. Our results demonstrate that the cell adhesion molecule CD34 was widely expressed by the MSCs of human major SGs, namely parotid (PAG), sublingual (SLG) and submandibular (SMG) glands. Further, gene expression analysis of CD34
cells derived from fetal SMGs showed significant upregulation of genes involved in cellular adhesion, proliferation, branching, extracellular matrix remodeling and organ development. Moreover, CD34
SMG cells exhibited elevated expression of genes encoding extracellular matrix, basement membrane proteins, and members of ERK, FGF and PDGF signaling pathways, which play key roles in glandular development, branching and homeostasis. In vitro CD34
cell derived SG-MSCs revealed multilineage differentiation potential. Intraglandular transplantation of cultured MSCs in immunodeficient mice led to their engraftment in the injected and uninjected contralateral and ipsilateral glands. Engrafted cells could be localized to the stroma surrounding acini and ducts. In summary, our data show that CD34
derived SG-MSCs could be a promising cell source for adoptive cell-based SG therapies, and bioengineering of artificial SGs.
Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment ...in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance.
In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.
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