mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase.

One critical aspect of cell proliferation is increased nucleotide synthesis, including pyrimidines. Pyrimidines are synthesized through de novo and salvage pathways. Prior studies established that the mammalian target of rapamycin complex 1 (mTORC1) promotes pyrimidine synthesis by activating the de novo pathway for cell proliferation.

Micronuclear collapse from oxidative damage.

Chromosome-containing micronuclei are a hallmark of aggressive cancers. Micronuclei frequently undergo irreversible collapse, exposing their enclosed chromatin to the cytosol. Micronuclear rupture catalyzes chromosomal rearrangements, epigenetic abnormalities, and inflammation, yet mechanisms safeguarding micronuclear integrity are poorly understood.

mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis.

Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction, and impairs mitophagy as evidenced by the accumulation of the PINK1 substrate pS65-Ubiquitin (pUb). We discovered MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes its active complex, resulting in increased rates of mitophagy.

How does the neuronal proteostasis network react to cellular cues?

Even though neurons are post-mitotic cells, they still engage in protein synthesis to uphold their cellular content balance, including for organelles, such as the endoplasmic reticulum or mitochondria. Additionally, they expend significant energy on tasks like neurotransmitter production and maintaining redox homeostasis. This cellular homeostasis is upheld through a delicate interplay between mRNA transcription-translation and protein degradative pathways, such as autophagy and proteasome degradation.

UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER). UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER.

Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron (MN) disease with severely limited treatment options. Identification of effective treatments has been limited in part by the lack of predictive animal models for complex human disorders. Here, we utilized pharmacologic ER stressors to exacerbate underlying sensitivities conferred by ALS patient genetics in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs).

Disruption of lysosomal proteolysis in astrocytes facilitates midbrain organoid proteostasis failure in an early-onset Parkinson's disease model.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate.

The CRL3 ubiquitin ligase-USP15 pathway governs the destruction of neurofilament proteins.

Giant axonal neuropathy (GAN) is caused by mutations in the gene encoding for gigaxonin (GIG), which functions as an adaptor of the CUL3-RBX1-GIG (CRL3) E3 ubiquitin ligase complex. The pathological hallmark of GAN is characterized by the accumulation of densely packed neurofilaments (NFs) in the axons. However, there are fundamental knowledge gaps in our understanding of the molecular mechanisms by which the ubiquitin-proteasome system controls the homeostasis of NF proteins.

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS.

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood.

Identifying E3 Ligase Substrates With Quantitative Degradation Proteomics.

Controlled protein degradation by the ubiquitin-proteasome pathway is critical for almost all cellular processes. E3 ubiquitin ligases are responsible for targeting proteins for ubiquitylation and subsequent proteasomal degradation with spatial and temporal precision. While studies have revealed various E3-substrate pairs involved in distinct biological processes, the complete substrate profiles of individual E3 ligases are largely unknown.

Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

Unlabelled: PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction and impairs mitophagy, driving accumulation of the PINK1 substrate pS65-Ubiquitin (pUb) in primary neurons and in vivo. We synthesized MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes an active heterocomplex, thereby increasing mitophagy.

Substitution of PINK1 Gly411 modulates substrate receptivity and turnover.

The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial.

Quantitative proteome remodeling characterization of two human reference pluripotent stem cell lines during neurogenesis and cardiomyogenesis.

Human pluripotent stem cells (PSCs) have become popular tools within the research community to study developmental and model diseases. While many induced-PSCs (iPSCs) from various genetic background sources are currently available, scientific advancement has been hampered by the considerable phenotypic variations observed between different iPSC lines. A recent collaborative effort selected a novel iPSC line to address this and encourage the adoption of a standardized iPSC line termed KOLF2.

Brain-derived autophagosome profiling reveals the engulfment of nucleoid-enriched mitochondrial fragments by basal autophagy in neurons.

Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of basal autophagy in the maintenance of neuronal health, we isolated autophagic vesicles from mouse brain tissue and used proteomics to identify the major cargos engulfed within autophagosomes, validating our findings in rodent primary and human iPSC-derived neurons. Mitochondrial proteins were identified as a major cargo in the absence of mitophagy adaptors such as OPTN.

Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1.

How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation–dependent diGLY sites in 22 proteins conserved across mouse and human systems.

Temporal proteomics during neurogenesis reveals large-scale proteome and organelle remodeling via selective autophagy.

Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis.

Super Heavy TMTpro Labeling Reagent: An Alternative and Higher-Charge-State-Amenable Stable-Isotope-Labeled TMTpro Variant.

Stable isotope labeling is a leading strategy for mass-spectrometry-based peptide quantification. Whereas TMTpro isobaric tagging can quantify up to 16 multiplexed samples in a single experiment, nonisobaric, yet chromatographically indistinguishable, variants of TMTpro reagents can be used in conjunction with the isobaric tag series for various peptide-targeting applications. Here we test the performance of two nonisobaric TMTpro variants, a stable-isotope-free TMTproZero tag and a nearly fully isotope-labeled "super-heavy" variant, shTMTpro, in a targeted assay for peptides of charge state 4+.

RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.

The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387.

The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase.

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins.