Biochemical and biophysical characterization of TyrA enzymes from symbiotic hyperthermophilic archaea Nanoarchaeum equitans and Ignicoccus hospitalis

The biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of both the trifunctional PD-CM-PDT from the smallest hyperthermophilc archaeon (Nanoarchaeum equitans) and the bifunctional CM-PD from its host (the crenarchaeon Ignicoccus hospitalis). Hexa histidine-tagged proteins were expressed in Escherichia coli and purified by chromatography on Ni-NTA affinity resin. Both enzymes were highly thermally stable and exhibited maximal activity at 90°C. CM, PD and PDT activities were detected at temperatures consistent with enzymes from extreme thermophiles. Kinetic analysis revealed that unlike most PDs, the two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP+ to NAD+ as a cofactor in the dehydrogenase reaction. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 μM) in a non-competitive fashion consistent with L-Phe’s combination at a site separate from that of prephenate. Gel filtration and analytical ultracentrifugation analysis of bifunctional CM-PD from I. hospitalis suggested that the enzyme is a native dimer. Limited proteolysis studies revealed that the enzyme is highly resistant to proteolysis but could be cleaved to yield a stable C-terminal PD domain. Mass spectrometry and mutagenesis studies confirmed that the PD domain of bifunctional I. hospitalis CM-PD could be independently isolated and expressed. Biochemical and biophysical characterization of this active truncated variant was performed and the results of solution studies were compared to those of the full-length protein and to information available from other PD enzymes. Guided by amino acid sequence alignment predictions and by models based on the available crystal structures of bacterial homologues, eight variants containing site-specific replacements were generated in I. hospitalis CM-PD as attempts to alter cofactor selectivity and substrate and end-product binding. Those variant proteins were kinetically characterized in order to help define the role of active site residues in substrate/ inhibitor interactions. These are the first studies exploring the aromatic amino acid biosynthetic pathway from the two archaeal organisms, which provide efficient and stable catalysts as excellent candidates for applications in biotechnology

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90

α-Synuclein Preformed Fibrils Bind to β-Neurexins and Impair β-Neurexin-Mediated Presynaptic Organization

Synucleinopathies form a group of neurodegenerative diseases defined by the misfolding and aggregation of α-synuclein (α-syn). Abnormal accumulation and spreading of α-syn aggregates lead to synapse dysfunction and neuronal cell death. Yet, little is known about the synaptic mechanisms underlying the α-syn pathology. Here we identified β-isoforms of neurexins (β-NRXs) as presynaptic organizing proteins that interact with α-syn preformed fibrils (α-syn PFFs), toxic α-syn aggregates, but not α-syn monomers. Our cell surface protein binding assays and surface plasmon resonance assays reveal that α-syn PFFs bind directly to β-NRXs through their N-terminal histidine-rich domain (HRD) at the nanomolar range (KD: ~500 nM monomer equivalent). Furthermore, our artificial synapse formation assays show that α-syn PFFs diminish excitatory and inhibitory presynaptic organization induced by a specific isoform of neuroligin 1 that binds only β-NRXs, but not α-isoforms of neurexins. Thus, our data suggest that α-syn PFFs interact with β-NRXs to inhibit β-NRX-mediated presynaptic organization, providing novel molecular insight into how α-syn PFFs induce synaptic pathology in synucleinopathies such as Parkinson’s disease and dementia with Lewy bodies

USP19 deubiquitinase inactivation regulates α-synuclein ubiquitination and inhibits accumulation of Lewy body-like aggregates in mice

Abstract The USP19 deubiquitinase is found in a locus associated with Parkinson’s Disease (PD), interacts with chaperonins, and promotes secretion of α-synuclein (α-syn) through the misfolding-associated protein secretion (MAPS) pathway. Since these processes might modulate the processing of α-syn aggregates in PD, we inactivated USP19 (KO) in mice expressing the A53T mutation of α-syn and in whom α-syn preformed fibrils (PFF) had been injected in the striatum. Compared to WT, KO brains showed decreased accumulation of phospho-synuclein (pSyn) positive aggregates. This improvement was associated with less activation of microglia and improved performance in a tail-suspension test. Exposure of primary neurons from WT and KO mice to PFF in vitro also led to decreased accumulation of pSyn aggregates. KO did not affect uptake of PFF nor propagation of aggregates in the cultured neurons. We conclude that USP19 instead modulates intracellular dynamics of aggregates. At an early time following PFF injection when the number of pSyn-positive neurons were similar in WT and KO brains, the KO neurons contained less aggregates. KO brain aggregates stained more intensely with anti-ubiquitin antibodies. Immunoprecipitation of soluble proteins from WT and KO brains with antibodies to pSyn showed higher levels of ubiquitinated oligomeric species in the KO samples. We propose that the improved pathology in USP19 KO brains may arise from decreased formation or enhanced clearance of the more ubiquitinated aggregates and/or enhanced disassembly towards more soluble oligomeric species. USP19 inhibition may represent a novel therapeutic approach that targets the intracellular dynamics of α-syn complexes

Additional file 1 of An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

Supplementary Material 1