Autophagy is a conserved lysosomal degradation pathway that degrades un-needed cellular components such as misfolded, aggregated, mutated and damaged proteins, organelles, and pathogens. Autophagy dysfunction is implicated in numerous diseases including neurodegenerative disorders, muscular diseases, cardiomyopathy, cancer and infectious diseases. Many proteins involved in autophagy contain intrinsically disordered regions (IDRs) that do not form stable secondary or tertiary structure. The structural flexibility of IDRs is thought to enable diverse and multiple interactions enabling them to regulate cell signaling pathways. Many IDRs have been shown to fold upon binding to ligands. BECN1, a key autophagy protein involved in autophagosome nucleation, contains two invariant CxxC motifs within a large BECN1 intrinsically disordered region (IDR) at the BECN1 N-terminus. The goal of the research was to uncover the functional roles of the invariant CxxC motifs which were hitherto not understood. The authors used inductively coupled plasma mass spectrometry to demonstrate that BECN1 binds Zn²⁺ in a 1:1 molar ratio, and that mutation of the invariant cysteines prevents co-ordination of Zn²⁺, demonstrating that the CxxC motifs are responsible for binding Zn²⁺. Zn^{2 …}

Semaglutide (SMG) is class of modified, acetylated peptide mimic commonly used as a commercial therapeutic to treat type-2 diabetes and obesity. Like other classes of peptide mimic therapeutics, SMG’s suffer from physical instabilities, including various aggregation and degradation pathways but also spontaneous emulsification into colloidal structures in the presence of certain hydrophobic surfaces, a process often termed “ouzo formation.” Researchers at the University of Delaware Center for Neutron Science, in collaboration with Eli Lilly, used a variety of biophysical methods including small-angle X-ray scattering (SAXS), circular dichroism (CD) and dynamic light scattering (DLS) to elucidate the fundamental physical mechanisms behind ouzo formation. SAXS experiments at BioCAT indicated that the colloidal droplets consisted of aggregated oligomers, which themselves form as a result of self-association of the peptides. Together with other experiments, the authors propose a mechanism where initial nucleation is catalyzed by adsorption of oligomers with a hydrophobic surface, where the effects of specific surfaces can be correlated with Hansen Solubility Parameters. Following nucleation (and a subsequent desorption step), nucleated droplets grow rapidly, ultimately resulting …

The ways in which starches, in particular digestion-resistant starches, are accommodated by gut bacteria remains relatively poorly understood at the molecular level. Digestion-resistant starches are accessed by specialized gut bacteria with specific carbohydrate-binding systems. One such system is the Ruminococcus bromii Starch Adherence System member 6 (Sas6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). The authors present a structural and functional characterization by crystallography, SAXS, native mass spec and other methods of one such system from Ruminococcus bromii (Sas6). The crystal structure of Sas6 showed that the RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. The overall structure was compact – both in crystals and in solution, as measured by size-exclusion chromatography-coupled SAXS (SEC-SAXS) – stabilized by significant hydrogen bonding networks between the domains. SEC-SAXS experiments …

Biomolecular condensates are involved in a range of cellular processes including stress response, protein degradation and gene expression. These condensates contain a wide range of unique macromolecules, but the drivers of this condensation, referred to as scaffolds, comprise only a very small fraction. The non-driver components are commonly referred to as ligands and may not phase separate on their own but nonetheless may help regulate assembly, disassembly and other material properties. One such ligand is ubiquitin (Ub) or its linked multimers (polyubiquitin chains), which are attached as posttranslational modifications to partner proteins and help determine various downstream signaling outcomes such as DNA repair. There is growing evidence that suggests the involvement of polyUb chains in phase separation acts as a mechanism for the reading and interpretation of said Ub code in the cell. Research from the Castaneda lab at Syracuse University has worked towards understanding the molecular rules by which polyUb chains …

Charcot Marie Tooth (CMT) disease is the most common form of heritable peripheral neuropathy, which are a group of inherited diseases affecting the peripheral nervous system (PNS). Myelin protein zero (MPZ) is necessary for normal myelin structural and function comprises ~50% of all proteins in the PNS; mutations in MPZ account for around 5% of CMT cases. MPZ is a transmembrane adhesion protein which holds together adjacent myelin membranes, thought to be mediated in part through homotypic interactions of its extracellular Ig domain. Exactly how the Ig domain of MPZ (Ig^MPZ) mediates adhesion of apposing membranes is not yet fully understood but is nonetheless important for understanding how myelin is constructed and how mutations in the Ig^MPZ cause disease. Models for how the Ig^MPZ might form oligomeric assemblies has been extrapolated from a protein crystal structure in which individual rat Ig^MPZ subunits packed together to form 3 weak interfaces involving Ig^MPZ organized tetramers, a ‘dimer’ interface linking tetramers together, and a hydrophobic interface that mediates binding to lipid …

Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. To address the need for better diagnostics, the authors sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms.

X-ray diffraction and myosin ATP turnover quantification assays show that patients with HFrEF-PH RV dysfunction with depressed isometric tension have reduced on state and disordered-relaxed (DRX) myosin; moreover, increasing the proportion of DRX myosin rescues the RV failure myocyte phenotype, whereas depressing DRX induces it. These results show, for the first time, that reduced basal DRX (on state) myosin contributes to a heart failure phenotype. HFrEF-PH RV myocytes exhibit blunted stretch-mediated recruitment of myosin from its super-relaxed to its DRX state, in association with depressed length-dependent active stiffness, a key contributor to Frank-Starling reserve. Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to …

Adaptor proteins are accessories to main proteins in signal transduction pathways that usually lack intrinsic enzymatic activity but instead facilitate the linking of binding partners together to enable the formation of larger signaling complexes. One widely expressed adaptor protein is the growth factor receptor-bound protein 2 (GRB2), which facilitates formation of cytoplasmic signaling complexes from a wide array of binding partners, including (among others) growth factor receptors, cytokine receptors and T cell receptor (TCR) ζ chains. As a consequence, the structure and function of GRB2 have become major areas of investigation for novel areas of interventions against various human diseases. GRB2 has been shown to exist in either a monomeric or dimeric state, where GRB2 dimers are formed by the exchange of protein segments between domains, a process termed “domain-swapping”. Prior to this work, swapping between the SH2 and SH3 domains of GRB2 has been demonstrated in the full-length structure, but SH2/SH2 domain-swapping had not. Researchers at the University of Iowa generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation …

Muscles can produce more force when stretched to a longer length at the same level of activating calcium, a poorly understood phenomenon known as myofilament length dependent activation (LDA). It was suggested several years ago that passive force generated by the giant elastic protein titin could be the length sensor behind this phenomenon, but direct evidence has been lacking. Investigators from the University of Muenster in Germany, Northern Arizona University, University of British Columbia, and the Illinois Institute of Technology used a mouse model with a cleavage site inserted into the titin protein allowing titin to be enzymatically cut in the mature tissue (with TEVp protease) to make it ineffective. Small-angle X-ray diffraction of muscle from these mice with and without cleavage showed that titin cleavage diminished the changes in length-dependent structural signatures (“priming”) associated with LDA. Strikingly, a titin-sensitive, length-dependent structural changes were also seen in thin filaments, which seems only possible if there are bridging structures between the thick and thin filaments in resting muscle, potentially comprised of myosin-binding protein C. These experiments firmly established titin as the length sensor in LDA and showed that LDA involves structural changes in both thick and thin filaments.

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Living cells are amazing little biochemical factories that conduct countless chemical reactions in a cellular soup packed with lipids, proteins, nucleic acids, and ions, keeping them all in their proper places at any given time. Cells maintain this organization even while carrying out complex tasks such as cell division, signaling, transcriptional regulation, and stress responses. One example of this is the careful management of stress granule formation, a process in which membraneless organelles transiently form to control the utilization of mRNA during stress. These granules form and disperse through reversible liquid-liquid phase transitions involving proteins and RNA in the granules. Recent research has demonstrated that RNA-binding proteins in these granules contain intrinsically disordered sequences, called prion-like low-complexity domains (PLCDs), that are critical to regulation of these reversible phase transitions. There is also mounting evidence that these transitions may be disrupted in neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS), in which mutations in PLCD-containing proteins, such as hnRNPA1, have been implicated as a cause of the disease. Recent …