Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in C. elegans

ABSTRACTAge-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in C. elegans; however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Further, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in “leaky” channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2-3 weeks in C. elegans, suggesting that reduced anti-oxidant capacity may contribute to the differences in life span amongst species.

Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients

A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic interrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes.

Repurposing Benzbromarone as Antifolate to Develop Novel Antifungal Therapy for Candida Albicans

Fungal infections in humans are responsible for mild to severe infections resulting in the systemic effects responsible for a large amount of mortality. The invasive fungal infections are having similar symptomatic effects to those of COVID-19. The COVID-19 patients are immunocompromised in nature and have a high probability of developing severe fungal infections resulting in the development of further complications. The existing antifungal therapy is having associated problems related to the development of drug resistance, sub-potent in nature, and the presence of undesirable toxic effects. The fungal dihydrofolate reductase is an essential enzyme involved in the absorption of dietary folic acid and its conversion into tetrahydrofolate, which is a coenzyme required for the biosynthesis of the fungal nucleotides. Thus, in the current study, an attempt has been made to identify potential folate inhibitors of Candida albicans by a computational drug repurposing approach. Benzbromarone is identified as a potential anti-folate agent based upon the molecular docking simulation-based virtual screening followed by the molecular dynamic simulation of the macromolecular complex for the development of a novel therapy for the treatment of candidiasis.

Conformational Changes in the Negative Arm of the Circadian Clock Correlate with Dynamic Interactomes Involved in Post-transcriptionally Regulated Processes

The circadian clock employs a transcriptional/translational negative feedback loop (TTFL) to anticipate environmental changes due to the Earth′s diurnal cycle, with regulation of organismal physiology believed to stem from temporal transcriptional activation by the positive arm. However, up to 80% of oscillating proteins do not have rhythmic mRNA, establishing circadian post-transcriptional regulation through unknown mechanisms. Given the pervasive conservation of the intrinsically disordered nature of negative-arm clock proteins, we hypothesized that post-transcriptional regulation may stem from conformational shifts in negative-arm proteins that time vacillations in the constituents of negative-arm macromolecular complexes to time cellular physiology. Our investigation of the negative arm clock protein in Neurospora crassa, FREQUENCY (FRQ), demonstrated temporal conformational fluidity correlated with daily changes in physiologically diverse macromolecular complex components. A parallel investigation of the macromolecular complexes centered around Drosophila melanogaster PERIOD (dPER) and human PERIOD (hPER2) found a similar number and physiological diversity of interacting partners in higher eukaryotes. Short linear motifs (SLiMs) associated with the interactors localized to disordered and phosphorylated regions on the PERs and FRQ, with disordered interactors oscillating in the macromolecular complexes over circadian time. This oscillation correlated with oscillations in post-transcriptionally regulated proteins, suggesting the negative arm may tune cellular physiology and proteostasis post-transcriptionally via vacillations in the circadian negative-arm macromolecular protein complexes.

Molecular Pathology of Sodium Channel Beta-Subunit Variants

The voltage-gated Na+ channel regulates the initiation and propagation of the action potential in excitable cells. The major cardiac isoform NaV1.5, encoded by SCN5A, comprises a monomer with four homologous repeats (I-IV) that each contain a voltage sensing domain (VSD) and pore domain. In native myocytes, NaV1.5 forms a macromolecular complex with NaVβ subunits and other regulatory proteins within the myocyte membrane to maintain normal cardiac function. Disturbance of the NaV complex may manifest as deadly cardiac arrhythmias. Although SCN5A has long been identified as a gene associated with familial atrial fibrillation (AF) and Brugada Syndrome (BrS), other genetic contributors remain poorly understood. Emerging evidence suggests that mutations in the non-covalently interacting NaVβ1 and NaVβ3 are linked to both AF and BrS. Here, we investigated the molecular pathologies of 8 variants in NaVβ1 and NaVβ3. Our results reveal that NaVβ1 and NaVβ3 variants contribute to AF and BrS disease phenotypes by modulating both NaV1.5 expression and gating properties. Most AF-linked variants in the NaVβ1 subunit do not alter the gating kinetics of the sodium channel, but rather modify the channel expression. In contrast, AF-related NaVβ3 variants directly affect channel gating, altering voltage-dependent activation and the time course of recovery from inactivation via the modulation of VSD activation.

Transcriptome Profiles of Leaves and Roots of Goldenrain Tree (Koelreuteria paniculata Laxm.) in Response to Cadmium Stress

Cadmium (Cd) pollution is a widespread environmental problem. In this study, we explored the transcriptome and biochemical responses of goldenrain tree (Koelreuteria paniculata Laxm.) leaves and roots to Cd stress. Leaf and root growth decreased substantially under Cd stress (50 mg/L CdCl2), but leaf and root antioxidant mechanisms were significantly activated. In RNA-seq analysis, roots treated with 25 mg/L CdCl2 featured enriched GO terms in cellular components related to intracellular ribonucleoprotein complex, ribonucleoprotein complex, and macromolecular complex. In leaves under Cd stress, most differentially expressed genes were enriched in the cellular component terms intrinsic component of membrane and membrane part. Weighted gene co-expression network analysis and analysis of module–trait relations revealed candidate genes associated with superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities and malondialdehyde (MDA). Ten transcription factors responded to Cd stress expression, including those in C2H2, MYB, WRKY, and bZIP families. Transcriptomic analysis of goldenrain tree revealed that Cd stress rapidly induced the intracellular ribonucleoprotein complex in the roots and the intrinsic component of membrane in the leaves. The results also indicate directions for further analyses of molecular mechanisms of Cd tolerance and accumulation in goldenrain tree.

Structures of the Cyanobacterial Phycobilisome

The phycobilisome is an elaborate antenna that is responsible for light-harvesting in cyanobacteria and red-algae. This large macromolecular complex captures incident sunlight and transfers the energy via a network of pigment molecules called bilins to the photosynthetic reaction centers. The phycobilisome of the model organism Synechocystis PCC 6803 consists of a core to which six rods are attached but its detailed molecular architecture and regulation in response to environmental conditions is not well understood. Here we present cryo-electron microscopy structures of the 6.2 MDa phycobilisome from Synechocystis PCC 6803 resolved at 2.1 Å (rods) to 2.7 Å (core), revealing three distinct conformations, two previously unknown. We found that two of the rods are mobile and can switch conformation within the complex, revealing a layer of regulation not described previously. In addition, we found a novel linker protein in the structure, that may represent a long-sought subunit that tethers the phycobilisome to the thylakoid membrane. Finally, we show how excitation energy is transferred within the phycobilisome and correlate our structures with known spectroscopic properties. Together, our results provide detailed insights into the biophysical underpinnings of cyanobacterial light harvesting and lay the foundation for bioengineering of future phycobilisome variants and artificial light harvesting systems.

Self-organization of kinetochore-fibers in human mitotic spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex

Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein–protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

RACK1 associates with RNA-binding proteins Vigilin and SERBP1 to control dengue virus replication

Dengue virus (DENV), a re-emerging virus transmitted by Aedes mosquitoes, causes severe pathogenesis in humans. No effective treatment is available against this virus. We recently identified the scaffold protein RACK1 as a component of the DENV replication complex, a macromolecular complex essential for viral genome amplification. Here, we show that RACK1 is important for DENV infection. RACK1 mediates DENV replication through binding to the 40S ribosomal subunit. Mass spectrometry analysis of RACK1 partners coupled to a loss-of-function screen identified the RNA binding proteins Vigilin and SERBP1 as DENV host dependency factors. Vigilin and SERBP1 interact with DENV viral RNA (vRNA), forming a ternary complex with RACK1 to mediate viral replication. Overall, our results indicate that RACK1 recruits Vigilin and SERBP1, linking the DENV vRNA to the translation machinery for optimal translation and replication.