Currently, our optimized strategy utilizes substrate-trapping mutagenesis and proximity-labeling mass spectrometry to provide quantitative analysis of protein complexes, encompassing those containing the protein tyrosine phosphatase PTP1B. This approach differs significantly from classical schemes by allowing for near-endogenous expression levels and escalating target enrichment stoichiometry without requiring the stimulation of supraphysiological tyrosine phosphorylation or the maintenance of substrate complexes during lysis and enrichment. Illustrative applications of this novel approach to PTP1B interaction networks in HER2-positive and Herceptin-resistant breast cancer models showcase its benefits. Through the use of cell-based models of HER2-positive breast cancer exhibiting either acquired or de novo Herceptin resistance, we have shown that PTP1B inhibitors significantly decreased both proliferation and cell viability. A differential analysis comparing substrate-trapping to wild-type PTP1B led to the identification of several novel protein targets of PTP1B, directly linked to HER2-stimulated signaling. The specificity of the method was internally validated by its concurrence with prior observations of substrate candidates. Integrating readily with evolving proximity-labeling platforms (TurboID, BioID2, etc.), this adaptable approach shows broad applicability across the PTP family to identify conditional substrate specificities and signaling nodes in disease models.
Both D1 receptor (D1R) and D2 receptor (D2R) expressing populations of spiny projection neurons (SPNs) in the striatum exhibit a high concentration of histamine H3 receptors (H3R). Studies on mice have revealed a cross-antagonistic interaction between the H3R and D1R receptors, observable at both the biochemical and behavioral levels. Interactive behavioral effects resulting from the concurrent stimulation of H3R and D2R receptors have been observed, however, the molecular underpinnings of this interaction remain poorly characterized. Treatment with the selective H3 receptor agonist R-(-),methylhistamine dihydrobromide attenuates the motor activity and repetitive behaviors brought about by D2 receptor agonists. Utilizing the proximity ligation assay, in conjunction with biochemical procedures, we found evidence of an H3R-D2R complex located in the mouse striatum. We also studied the consequences of the combination of H3R and D2R agonism on the phosphorylation levels of several signaling molecules by employing immunohistochemical techniques. Phosphorylation of mitogen- and stress-activated protein kinase 1, as well as rpS6 (ribosomal protein S6), displayed little to no change in these conditions. Given the involvement of Akt-glycogen synthase kinase 3 beta signaling pathways in various neuropsychiatric conditions, this research could illuminate how H3R influences D2R function, thereby improving our comprehension of the pathophysiological mechanisms associated with histamine-dopamine interactions.
The brain pathology shared by synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is the buildup of misfolded alpha-synuclein (α-syn) protein. Tenapanor PD patients carrying hereditary -syn mutations are more prone to an earlier age of disease onset and more severe clinical presentations than their sporadic PD counterparts. 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. Tenapanor A cryo-electron microscopy structure of α-synuclein fibrils with the hereditary A53E mutation is presented, achieved at 338 Å resolution. Tenapanor In terms of structure, the A53E fibril, akin to fibrils from wild-type and mutant α-synuclein, is made up of two symmetrically placed protofilaments. This structure of synuclein fibrils is unprecedented, showing differences from all other known structures, not just at the proto-filament boundaries, but also among the packed residues located within the same proto-filaments. Among all -syn fibrils, the A53E fibril exhibits the smallest interface and least buried surface area, due to only two contacting residues. A53E's structural variation and residue re-arrangement within the same protofilament is notable, particularly at a cavity near its fibril core. Compared to wild-type and mutants such as A53T and H50Q, A53E fibrils exhibit a slower fibrillization rate and decreased stability, yet evidence strong seeding capabilities in alpha-synuclein biosensor cells and primary neurons. Essentially, our study proposes to showcase the structural divergences, both within and between the protofilaments of A53E fibrils, to interpret the fibril assembly and cellular seeding of α-synuclein pathology in disease, advancing our knowledge of the structure-function relationship of α-synuclein mutants.
Organismal development relies on MOV10, an RNA helicase, which displays robust expression in the postnatal brain. AGO2-mediated silencing is contingent upon MOV10, a protein that is also associated with AGO2. The miRNA pathway's fundamental action is undertaken by AGO2. MOV10's ubiquitination, resulting in its breakdown and detachment from the messenger RNA it is bound to, has been observed. Despite this, no other post-translational modifications possessing functional relevance have been detailed. Employing mass spectrometry, we identified MOV10 phosphorylation at serine 970 (S970) on the C-terminal end of the protein within the cellular environment. By changing serine 970 to a phospho-mimic aspartic acid (S970D), the unfolding of the RNA G-quadruplex was impeded, exhibiting a similar pattern to the disruption caused by the mutation in the helicase domain (K531A). Alternatively, the S970A substitution within MOV10 produced the unfolding of the modeled RNA G-quadruplex. In our RNA-seq analysis of S970D's cellular role, we found decreased expression of MOV10-enhanced Cross-Linking Immunoprecipitation targets compared to WT controls. The introduction of S970A resulted in an intermediate effect, signifying that S970 plays a protective role in the mRNAs. In complete cell extracts, MOV10 and its variants displayed similar binding to AGO2; however, silencing AGO2 prevented the mRNA degradation induced by S970D. Consequently, MOV10's activity safeguards mRNA from AGO2's influence; the phosphorylation of serine 970 diminishes this protective effect, thereby leading to AGO2-driven mRNA degradation. 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. Ultimately, our data indicates that MOV10 phosphorylation allows for the interaction of AGO2 with the 3' untranslated region of translating mRNAs, causing their degradation.
The application of powerful computational methods is profoundly altering protein science, with particular emphasis on structure prediction, where AlphaFold2 is adept at predicting a vast number of natural protein structures from their corresponding sequences, while other artificial intelligence techniques enable the development of new structures from first principles. These methods raise the crucial question: how profoundly do we understand the sequence-to-structure/function linkages they are purportedly capturing? The current view of one protein assembly type, the -helical coiled coils, is provided in this perspective. These sequences, consisting of straightforward repetitions of hydrophobic (h) and polar (p) residues, (hpphppp)n, are critical in determining the folding and aggregation of amphipathic helices into bundles. Although numerous bundle configurations are feasible, these bundles can consist of two or more helices (different oligomers); the helices can exhibit parallel, antiparallel, or a combination of orientations (varying topologies); and the helical sequences can be identical (homomeric) or distinct (heteromeric). Consequently, the sequence-to-structure correspondences within the hpphppp repetitions are crucial for discerning these states. This problem is investigated through a three-level analysis; physics' parametric methodology generates a variety of potential coiled-coil backbone structures, first. Chemistry's second function is to investigate and articulate the connection between sequence and structure. From a biological perspective, the tailored and functional roles of coiled coils inspire the use of these structures in synthetic biology applications, third. Although the chemical underpinnings are well-understood, and significant progress has been made in physics, the precise prediction of the relative stability of different coiled-coil conformations still represents a major hurdle. However, a wealth of opportunities for discovery still lie in the biological and synthetic study of these structures.
The decision for apoptotic cell death is made at the mitochondria, a location where BCL-2 family proteins function to regulate this crucial process. BIK, a resident protein of the endoplasmic reticulum, acts to inhibit the mitochondrial BCL-2 proteins, thereby promoting the process of apoptosis. This paper, by Osterlund et al. and published recently in the JBC, focused on this intricate problem. To their surprise, the endoplasmic reticulum and mitochondrial proteins were seen to travel towards each other and meet at the connection site of the two organelles, constructing a 'bridge to death'.
Prolonged torpor is a common characteristic of numerous small mammals during winter hibernation. The non-hibernation season finds them as a homeotherm, but the hibernation season marks a change to a heterothermic state. The hibernation cycle of Tamias asiaticus chipmunks involves alternating periods of deep torpor, lasting 5 to 6 days, with a body temperature (Tb) between 5 and 7°C. Subsequent arousal episodes, lasting 20 hours, restore normothermic Tb levels. Our study focused on liver Per2 expression to understand the regulation of the peripheral circadian clock in a mammal that hibernates.