A Glance into MTHFR Deficiency at a Molecular Level

MTHFR deficiency still deserves an investigation to associate the phenotype to protein structure variations. To this aim, considering the MTHFR wild type protein structure, with a catalytic and a regulatory domain and taking advantage of state-of-the-art computational tools, we explore the properties of 72 missense variations known to be disease associated. By computing the thermodynamic ΔΔG change according to a consensus method that we recently introduced, we find that 61% of the disease-related variations destabilize the protein, are present both in the catalytic and regulatory domain and correspond to known biochemical deficiencies. The propensity of solvent accessible residues to be involved in protein-protein interaction sites indicates that most of the interacting residues are located in the regulatory domain, and that only three of them, located at the interface of the functional protein homodimer, are both disease-related and destabilizing. Finally, we compute the protein architecture with Hidden Markov Models, one from Pfam for the catalytic domain and the second computed in house for the regulatory domain. We show that patterns of disease-associated, physicochemical variation types, both in the catalytic and regulatory domains, are unique for the MTHFR deficiency when mapped into the protein architecture.

Neurotoxin-mediated ­­potent activation of the axon degeneration regulator SARM1

Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the nicotinamide adenine dinucleotide (NAD)-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet know is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.

Septin-microtubule association via a motif unique to the isoform 1 of septin 9 tunes stress fibers

Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies.

AMPK promotes Arf6 activation in a kinase-independent manner upon energy deprivation

AMP-activated protein kinase (AMPK) is a crucial cellular nutrient and energy sensor that maintains energy homeostasis. AMPK also governs cancer cell invasion and migration by regulating gene expression and activating multiple cellular signaling pathways. ADP-ribosylation factor 6 (Arf6) can be activated via nucleotide exchange by guanine nucleotide exchange factors (GEFs), and its activation also regulates tumor invasion and migration. By studying GEF-mediated Arf6 activation, we elucidated that AMPK functions as a noncanonical GEF for Arf6 in a kinase-independent manner. Moreover, by examining the physiological role of the AMPK-Arf6 axis, we determined that AMPK activates Arf6 upon glucose starvation and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) treatment. We further identified the binding motif in the C-terminal regulatory domain of AMPK that is responsible for promoting Arf6 activation and thus inducing cell migration and invasion. These findings reveal a noncanonical role of AMPK in which its C-terminal regulatory domain serves as a GEF for Arf6 during energy deprivation.

Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex

Photoprotection is an essential mechanism in photosynthetic organisms to balance the harvesting of light energy against the risks of photodamage. In cyanobacteria, photoprotective non-photochemical quenching relies on the interaction between a photoreceptor, the Orange Carotenoid Protein (OCP), and the antenna, the phycobilisome (PBS). Here we report the first structure of the OCP-PBS complex at 2.7 Å overall resolution obtained by cryo-electron microscopy. The structure shows that the 6.2 MDa PBS is quenched by four 34 kDa OCP organized as two dimers. The complex also reveals that the structure of the active form of the OCP is drastically different than its resting, non-quenching form, with an ~60 Å displacement of its regulatory domain. These results provide a high-resolution blueprint of the structural basis of the protective quenching of excess excitation energy that enables cyanobacteria to harvest light energy and fix CO2 across environmentally diverse and dynamic surface of our planet.

PRD-Containing Virulence Regulators (PCVRs) in Pathogenic Bacteria

Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved pathway in bacteria that couples transport of sugars with phosphorylation to monitor host carbohydrate availability. A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Streptococcus pyogenes Mga and Bacillus anthracis AtxA. These paradigm PCVRs undergo phosphorylation, potentially via the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from Streptococcus pneumoniae (MgaSpn) and Enterococcus faecalis (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation via potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.

PKC-e Regulates Vesicle Delivery and Focal Exocytosis for Efficient IgG-mediated Phagocytosis

PKC-e is required for membrane addition during IgG-mediated phagocytosis; its role in this process is ill-defined. High resolution imaging revealed that PKC-e exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-e colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-e and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles are not delivered in macrophages from PKC-e null mice, or upon dissociation of the Golgi-associated pool of PKC-e, implicates Golgi-tethered PKC-e as a driver of Golgi-to-phagosome trafficking. Finally, we established that PKC-e's regulatory domain is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-e in focal exocytosis: its regulatory domain drives Golgi-derived vesicles to the phagosome while catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.

Single nucleus pituitary transcriptomic and epigenetic landscape reveals human stem cell heterogeneity with diverse regulatory mechanisms

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility for GATA3, but with TF expression for POMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.