An improved approach, optimized for our needs, now utilizes substrate-trapping mutagenesis coupled with proximity-labeling mass spectrometry to quantitatively examine protein complexes containing the protein tyrosine phosphatase PTP1B. In contrast to traditional methodologies, this approach enables near-endogenous expression levels and a rising stoichiometry of target enrichment, dispensing with the requirement for supraphysiological tyrosine phosphorylation stimulation or the preservation of substrate complexes throughout lysis and enrichment processes. This new approach's strengths are evident when investigating PTP1B interaction networks in models of both HER2-positive and Herceptin-resistant breast cancer. In HER2-positive breast cancer, cell-based models of both acquired and de novo Herceptin resistance displayed decreased proliferation and viability when exposed to PTP1B inhibitors, as our study has revealed. By way of differential analysis, we contrasted substrate-trapping with the wild-type PTP1B, revealing multiple novel protein targets of PTP1B with a key role in HER2-induced signaling. Internal validation for the method's specificity was provided by corroborating the results with earlier reports of substrate candidates. Evolving proximity-labeling platforms (TurboID, BioID2, etc.) are readily compatible with this flexible strategy, which has broad applicability across the entire PTP family to identify conditional substrate specificities and signaling nodes in human disease models.

The striatum's D1 receptor (D1R) and D2 receptor (D2R) expressing spiny projection neurons (SPNs) display a high level of histamine H3 receptor (H3R) enrichment. The presence of a cross-antagonistic interaction between H3R and D1R receptors in mice has been corroborated by both behavioral and biochemical findings. Despite the described interactive behavioral effects associated with the co-activation of H3R and D2R receptors, the molecular mechanisms mediating this phenomenon remain poorly understood. We observed that the activation of H3 receptors, specifically by the selective agonist R-(-),methylhistamine dihydrobromide, reduces the motor activity and stereotypies induced by D2 receptor agonists. Through biochemical investigations and the use of the proximity ligation assay, we observed an H3R-D2R complex within the mouse striatum's structure. In parallel, we analyzed the effects of simultaneous H3R and D2R activation on the phosphorylation levels of several signaling proteins employing immunohistochemistry. Mitogen- and stress-activated protein kinase 1 and rpS6 (ribosomal protein S6) phosphorylation levels exhibited minimal alteration under these experimental circumstances. Acknowledging the involvement of Akt-glycogen synthase kinase 3 beta signaling in several neuropsychiatric disorders, this research may help delineate the role of H3R in modulating D2R activity, ultimately promoting a better comprehension of the underlying pathophysiology associated with the interaction between the histamine and dopamine systems.

In synucleinopathies, exemplified by Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), the presence of misfolded alpha-synuclein protein (-syn) accumulated in the brain is a defining characteristic. Opaganib SPHK inhibitor Individuals with Parkinson's Disease (PD) harboring hereditary -syn mutations often experience an earlier disease onset and more severe clinical manifestations compared to those with sporadic PD. Hence, uncovering the impact of hereditary mutations on the arrangement of alpha-synuclein fibrils offers a pathway to understanding the structural foundation of these synucleinopathies. Opaganib SPHK inhibitor At a resolution of 338 Ångströms, this cryo-electron microscopy study reveals the structure of α-synuclein fibrils, which harbor the hereditary A53E mutation. Opaganib SPHK inhibitor The symmetry of the A53E fibril, composed of two protofilaments, mirrors the structure of the fibrils found in wild-type and mutant α-synuclein. The arrangement of the new synuclein fibrils is distinct from existing structures, deviating not only at the connecting points between proto-filaments, but also among the tightly-packed residues internal to each proto-filament. Among all -syn fibrils, the A53E fibril exhibits the smallest interface and least buried surface area, due to only two contacting residues. The residue rearrangements and variations in structure of A53E, found within the same protofilament, are distinct, situated near the fibril core's cavity. A53E fibrils, in contrast to the wild-type and other variants like A53T and H50Q, display a slower fibrillization rate and lower stability, while also demonstrating significant seeding within alpha-synuclein biosensor cells and primary neurons. Crucially, our research intends to accentuate the structural diversities within and between the protofilaments of A53E fibrils, while simultaneously interpreting fibril development and cellular seeding of α-synuclein pathology in disease, ultimately contributing to our comprehension of the structure-function relationship of mutated α-synuclein.

For organismal development, MOV10, an RNA helicase, shows significant expression in the postnatal brain. Essential for AGO2-mediated silencing, MOV10 is also an AGO2-associated protein. The miRNA pathway's fundamental action is undertaken by AGO2. MOV10, marked by ubiquitination, leads to its degradation and dissociation from bound messenger RNA. No other functionally consequential post-translational modifications have been characterized. Mass spectrometry confirms the cellular phosphorylation of MOV10 at serine 970 (S970) within the C-terminus of the protein. Altering serine 970 to a phospho-mimic aspartic acid (S970D) prevented the unfolding of the RNA G-quadruplex, mirroring the effect of the mutation in the helicase domain (K531A). Instead of stabilizing, the alanine substitution at position 970 (S970A) within MOV10 caused the model RNA G-quadruplex structure to unravel. Using RNA-seq, we observed that the S970D substitution led to a decrease in the expression of genes targeted by MOV10, as revealed through crosslinking immunoprecipitation, relative to the wild-type control. The effect on mRNA expression suggests a potential protective role of S970 in these targets. Whole-cell extracts revealed comparable binding of MOV10 and its substitutions to AGO2; however, AGO2 knockdown eliminated the mRNA degradation effect triggered by S970D. Therefore, the activity of MOV10 shields mRNA from AGO2's targeting; S970 phosphorylation hinders this shielding, consequently facilitating AGO2-mediated mRNA breakdown. S970's C-terminal placement relative to the MOV10-AGO2 interaction site brings it near a disordered region, possibly affecting the phosphorylation-dependent interaction between AGO2 and target messenger ribonucleic acids. The evidence presented highlights how MOV10 phosphorylation enables the interaction of AGO2 with the 3' untranslated regions of translating mRNAs, thereby inducing their degradation.

The field of protein science is undergoing a transformation, driven by powerful computational methods dedicated to structure prediction and design. AlphaFold2, for instance, accurately predicts a variety of natural protein structures from their sequences, and other AI methodologies are now capable of designing new protein structures from the ground up. The methods' capture of sequence-to-structure/function relationships naturally leads to the question: to what degree do we understand the underlying principles these methods reveal? Our current comprehension of -helical coiled coils, a specific protein assembly class, is elucidated by this perspective. From a superficial perspective, the sequences (hpphppp)n, composed of repeating hydrophobic (h) and polar (p) residues, are fundamental to the folding and bundling of amphipathic helices. Nonetheless, a multitude of distinct bundles are conceivable, featuring two or more helices (representing various oligomeric states); the helices may exhibit parallel, antiparallel, or a combination of these orientations (diverse topological arrangements); and the helical sequences can be identical (homomeric) or divergent (heteromeric). Consequently, the interplay of sequence and structure within the repeating hpphppp motifs is needed to distinguish these states. From a threefold perspective, I begin by exploring current knowledge of this issue; physics provides a parametric basis for generating the multitude of potential coiled-coil backbone configurations. Chemistry's second function is to investigate and articulate the connection between sequence and structure. Third, nature's utilization of coiled coils, as evident in biological systems, provides a blueprint for their applications within synthetic biology. While the chemistry of coiled coils is largely understood and physical models are partially successful, the predictive capability for relative stability of different coiled-coil forms remains a significant hurdle. Further opportunities for discovery, however, are available in the domains of biology and synthetic biology of coiled coils.

The commitment to programmed cell death via apoptosis is initiated at the mitochondria, with the BCL-2 protein family playing a regulatory role within this subcellular compartment. Resident protein BIK, found in the endoplasmic reticulum, prevents mitochondrial BCL-2 proteins from functioning, thus initiating the process of apoptosis. The Journal of Biological Chemistry recently featured Osterlund et al.'s investigation into this challenging issue. Intriguingly, the movement of endoplasmic reticulum and mitochondrial proteins towards each other, and their meeting at the contact site between the two organelles, resulted in a 'bridge to death'.

A multitude of small mammals experience a period of prolonged torpor during winter hibernation. The homeotherm nature of the creature is observed in the non-hibernation season, changing to a heterothermic nature during hibernation. In the hibernation season, chipmunks of the species Tamias asiaticus experience periods of profound torpor lasting 5 to 6 days, during which their body temperature (Tb) drops to 5-7°C. Between these episodes, 20-hour arousal periods raise their Tb to the normal range. Our study focused on liver Per2 expression to understand the regulation of the peripheral circadian clock in a mammal that hibernates.