The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies.

Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.

Identification and functional characterization of the nuclear and nucleolar localization signals in the intrinsically disordered region of nucleomethylin.

The nucleolar localization of proteins is regulated by specific signals directing their trafficking to nucleus and nucleolus. Here, we elucidate the mechanism underlying the nuclear and nucleolar localization of the nucleomethylin (NML) protein, focusing on its nuclear localization signals (NLSs) and nucleolar localization signal (NoLS). Using a combination of bioinformatic analysis and experimental validation, we identified two monopartite and one bipartite NLS motifs within NML. The combined presence of both monopartite NLSs significantly enhances nuclear localization of the protein, while specific basic amino acid clusters within the bipartite NLS are crucial for their functionality. We also reveal the functional role of the NLS-coupled NoLS motif in driving nucleolar localization of NML, which contains an arginine-rich motif essential for its function. The basic residues of the arginine-rich motif of NoLS of NML interacts with nucleophosmin 1 (NPM1), allowing the possible liquid-liquid phase separation and retention of NML in the nucleolus. Remarkably, the strong NoLS of NML can direct the nucleolar localization of a cytosolic protein, aldolase, emphasizing its potency. Overall, our findings provide insights into the combinatorial functioning of NLSs and NoLS in regulating the subcellular localization of NML, highlighting the intricate regulatory mechanisms governing its localization within the nucleus and nucleolus.

Assessing type I collagen expression and quality in cellular models of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of genetic disorders of bone formation characterized by soft and shorter brittle bones in affected individuals. OI is generally considered a collagenopathy resulting from abnormal expression of type I collagen. As assay system to detect the cellular level and quality of type I collagen would help in rapid and correct detection of OI from the diagnostic perspectives. Here, we report an immunofluorescence assay for detection of type I collagen in fibroblast models of OI and represented them into two broad categories based on the expression level and aggregation characteristics of pro-α1(I). Cell phenotypic assays of pro-α1(I) in OI-related gene knocked down fibroblasts revealed aggregates of pro-α1(I) in conditions with knockdown of SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, TMEM38B, MESD, and KDELR2, whereas pro-α1(I) expression was very low in fibroblasts which had knockdown of IFITM5, SP7, BMP1, WNT1, CREB3L1, MBTPS2, and CCDC134. The expression of pro-α1(I) showed abundant and non-aggregated distribution in the fibroblasts with knockdown of non-OI skeletal disorder-related genes (RAB33B and IFT52). The in vitro assay accurately detected abnormally expressed pro-α1(I) levels in cellular models of various types of OI. Thus, this procedure represents a promising point-of-detection assay for potential diagnosis and therapeutic decisions in OI.

Nuclear proteostasis imbalance in laminopathy-associated premature aging diseases.

Laminopathies are a group of rare genetic disorders with heterogeneous clinical phenotypes such as premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, and so on. The cellular phenomena associated with laminopathy invariably show disruption of nucleoskeleton of lamina due to deregulated expression, localization, function, and interaction of mutant lamin proteins. Impaired spatial and temporal tethering of lamin proteins to the lamina or nucleoplasmic aggregation of lamins are the primary molecular events that can trigger nuclear proteotoxicity by modulating differential protein-protein interactions, sequestering quality control proteins, and initiating a cascade of abnormal post-translational modifications. Clearly, laminopathic cells exhibit moderate to high nuclear proteotoxicity, raising the question of whether an imbalance in nuclear proteostasis is involved in laminopathic diseases, particularly in diseases of early aging such as HGPS and laminopathy-associated premature aging. Here, we review nuclear proteostasis and its deregulation in the context of lamin proteins and laminopathies.

Genome sequencing identifies a large non-coding region deletion of SNX10 causing autosomal recessive osteopetrosis.

Autosomal recessive osteopetrosis (ARO) is a rare genetic disorder caused by impaired osteoclast activity. In this study, we describe a 4-year-old boy with increased bone density due to osteopetrosis, autosomal recessive 8. Using genome sequencing, we identified a large deletion in the 5'-untranslated region (UTR) of SNX10 (sorting nexin 10), where the regulatory region of this gene is located. This large deletion resulted in the absence of the SNX10 transcript and led to abnormal osteoclast activity. SNX10 is one of the nine genes known to cause ARO, shown to interact with V-ATPase (vacuolar type H( + )-ATPase), as it plays an important role in bone resorption. Our study highlights the importance of regulatory regions in the 5'-UTR of SNX10 for its expression while also demonstrating the importance of genome sequencing for detecting large deletion of the regulatory region of SNX10.

An IRES-dependent translation of HYPK mRNA generates a truncated isoform of the protein that lacks the nuclear localization and functional ability.

Different mechanisms of translation initiation process exist to start the protein synthesis from various viral and eukaryotic mRNA. The cap-independent and tertiary structure directed translation initiation of mRNAs forms the basis of internal ribosome entry site (IRES) mediated translation initiation that helps in cellular protein production in different conditions. HYPK protein sequesters different aggregation-prone proteins to help in the cellular proteostasis. HYPK mRNA is differentially translated from an internal start/initiation codon to generate an amino terminal-truncated isoform (HSPC136) of HYPK protein. In this study, we report that an IRES-dependent translation initiation of HYPK mRNA results in the formation of the HSPC136/HYPK-ΔN isoform of HYPK protein. The IRES-driven translation product, HYPK-ΔN, lacks the N-terminal tri-arginine motif that acts as the nuclear localization signal (NLS) in the full-length HYPK protein. While the full-length HYPK protein translocates to the nucleus and prevents the aggregation of the mutant p53 (p53-R248Q) protein, the HYPK-ΔN lacks this activity. The NLS of HYPK is not evolutionarily conserved and its exclusive presence in the HYPK of higher eukaryotic animals imparts additional advantage to the HYPK protein in tackling the cytosolic as well as nuclear protein aggregates. The presence of the NLS in full-length HYPK also allows this protein to modulate the cell cycle. These results provide a mechanistic detail of HYPK mRNA's translation initiation control by an IRES that dictates the formation of HYPC136/HYPK-ΔN which lacks the nuclear localization and functional ability.

Disordered Nanostructure in Huntingtin Interacting Protein K Acts as a Stabilizing Switch To Prevent Protein Aggregation.

Protein misfolding due to mutation(s) and/or generation of unstable intermediate state(s) can be the cause of aberrant aggregations, leading to cellular degeneration. While molecular signatures like amyloidogenic regions cause aggregation, other features in proteins, like disorder and unique complexity regions, regulate and restrict such adhesive accumulation processes. Huntingtin interacting protein K (HYPK) is an aggregation-prone protein. Using various biophysical, microscopy, and computational techniques, we have deciphered how HYPK's N-terminal nanodisordered region plays a significant modulatory role in preventing its own aggregation and that of other proteins. HYPK's C-terminal hydrophobic regions lead to annular oligomerization and intermolecular charge interactions among the residues of low-complexity region (LCR) generate amorphous aggregates. The N-terminal disordered nanostructure loops toward the C-terminus, and a negative charge-rich patch in this region interacts with the LCR to shield LCR's positive charges. This interaction is required to prevent HYPK aggregation. Loss of this interaction causes partial unfolding of the structured C-terminus, resulting in HYPK's molten globule-like state and rapid annular oligomerization. The N-terminus also determines the specificity to mediate the differential bindings with aggregation-prone and wild type Huntingtin-exon1 proteins (Huntingtin97Q-exon1 and Huntingtin25Q-exon1). A sliding interaction of the specific N-terminal segment of HYPK along the extended polyglutamine region of Huntingtin-exon1 is responsible for HYPK's higher affinity for aggregation-prone Huntingtin than for its non-aggregating counterpart. Overall, our study provides evidence of the existence of disordered nanostructure in HYPK protein that mechanistically plays a decisive role in preventing both self and non-self protein aggregation.

Membrane tension sensing formin-binding protein 1 is a neuronal nutrient stress-responsive Golgiphagy receptor.

BACKGROUND: Nutrient stress-responsive neuronal homeostasis relies on intricate autophagic mechanisms that modulate various organelle integrity and function. The selective autophagy of the Golgi, known as Golgiphagy, regulates secretory processes by modulating vesicle trafficking during nutrient starvation. RESULTS: In this study, we explored a genetic screen of BAR-domain-containing proteins to elucidate the role of formin-binding protein 1 (FNBP1) as a Golgiphagy receptor in modulating Golgi dynamics in response to varying nutrient availability in neurons. Mapping the systems network of FNBP1 and its interacting proteins reveals the putative involvement of FNBP1 in autophagy and Golgi-associated processes. While nutrient depletion causes Golgi fragmentation, FNBP1 preferentially localizes to the fragmented Golgi membrane through its 284FEDYTQ289 motif during nutrient stress. Simultaneously, FNBP1 engages in molecular interactions with LC3B through a conserved 131WKQL134 LC3 interacting region, thereby sequestering the fragmented Golgi membrane in neuronal autophagosomes. Increased aggregation of GM130, abnormal clumping of RAB11-positive secretory granules, and enhanced senescent death of FNBP1-depleted starved neurons indicate disruptions of neuronal homeostasis under metabolic stress. CONCLUSION: The identification of FNBP1 as a nutrient stress-responsive Golgiphagy receptor expands our insights into the molecular mechanisms underlying Golgiphagy, establishing the crosstalk between nutrient sensing and membrane tension-sensing regulatory autophagic processes of Golgi turnover in neurons.

Cadmium toxicity on endoplasmic reticulum functioning.

Cadmium (Cd) is a heavy metal pollutant widely distributed in the environment due to industrial activities, mining, and agricultural practices. Cadmium-induced Toxicity exerts profound effects on ER functioning through multiple mechanisms, leading to cellular dysfunction and pathological consequences. Cadmium disrupts protein folding and activates the unfolded protein response (UPR). Cd exposure leads to the accumulation of misfolded proteins, triggering UPR pathways mediated by critical ER transmembrane sensors: IRE1, PERK, and ATF6. The subsequent UPR aims to restore ER homeostasis but can also induce apoptosis under severe stress conditions. Cd disrupts ER calcium homeostasis by inhibiting the SERCA pump, further exacerbating ER stress. The generation of reactive oxygen species (ROS also plays a critical role in Cd toxicity, damaging ER-resident proteins and amplifying UPR activation). Cadmium also affects the lipid metabolism. This review examines the mechanisms by which Cd toxicity impairs ER functioning, disruption of protein folding and quality control mechanisms, and dysregulation of calcium signaling and lipid metabolism. The subsequent cellular consequences, including oxidative stress, apoptosis, and inflammation, are discussed in the context of Cd-induced pathogenesis of diseases such as Cancer and neurodegenerative and cardiovascular disorders. Finally, potential therapeutic strategies must be explored to mitigate the adverse effects of Cd on ER functioning and human health.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Acyl-CoA binding protein regulates nutrient-dependent autophagy.

BACKGROUND: Homeostasis of autophagy under normal conditions and nutrient stress is maintained by adaptive activation of regulatory proteins. However, the protein-lipid crosstalk that modulates the switch from suppression to activation of autophagy initiation is largely unknown. RESULTS: Here, we show that human diazepam-binding inhibitor (DBI), also known as acyl-CoA binding protein (ACBP), binds to phosphatidylethanolamine of the phagophore membrane under nutrient-rich growth conditions, leading to inhibition of LC3 lipidation and suppression of autophagy initiation. Specific residues, including the conserved tyrosine residues of DBI, interact with phosphatidylethanolamine to stabilize the later molecule in the acyl-CoA binding cavity of the protein. Under starvation, phosphorylation of serine-21 of DBI mediated by the AMP-activated protein kinase results in a drastic reduction in the affinity of the protein for phosphatidylethanolamine. The release of serine-21 phosphorylated DBI from the phagophore upon nutrient starvation restores the high LC3 lipidation flux and maturation of the phagophore to autophagosome. CONCLUSION: DBI acts as a strategic barrier against overactivation of phagophore maturation under nutrient-rich conditions, while triggering autophagy under nutrient-deficient conditions.

Mutant MESD links cellular stress to type I collagen aggregation in osteogenesis imperfecta type XX.

Aberrant forms of endoplasmic reticulum (ER)-resident chaperones are implicated in loss of protein quality control in rare diseases. Here we report a novel mutation (p.Asp233Asn) in the ER retention signal of MESD by whole exome sequencing of an individual diagnosed with osteogenesis imperfecta (OI) type XX. While MESDD233N has similar stability and chaperone activity as wild-type MESD, its mislocalization to cytoplasm leads to imbalance of ER proteostasis, resulting in improper folding and aggregation of proteins, including LRP5 and type I collagen. Aggregated LRP5 loses its plasma membrane localization to disrupt the expression of WNT-responsive genes, such as BMP2, BMP4, in proband fibroblasts. We show that MESD is a direct chaperone of pro-α1(I) [COL1A1], and absence of MESDD233N in ER results in cytosolic type I collagen aggregates that remain mostly not secreted. While cytosolic type I collagen aggregates block the intercellular nanotubes, decreased extracellular type I collagen also results in loss of interaction of ITGB1 with type I collagen and weaker attachment of fibroblasts to matrix. Although proband fibroblasts show increased autophagy to degrade the aggregated type I collagen, an overall cellular stress overwhelms the proband fibroblasts. In summary, we present an essential chaperone function of MESD for LRP5 and type I collagen and demonstrating how the D233N mutation in MESD correlates with impaired WNT signaling and proteostasis in OI.

Deep intronic mutation in CRTAP results in unstable isoforms of the protein to induce type I collagen aggregation in a lethal type of osteogenesis imperfecta type VII.

Genetic mutations are involved in Mendelian disorders. Unbuffered intronic mutations in gene variants can generate aberrant splice sites in mutant transcripts, resulting in mutant isoforms of proteins with modulated expression, stability, and function in diseased cells. Here, we identify a deep intronic variant, c.794_1403A>G, in CRTAP by genome sequencing of a male fetus with osteogenesis imperfecta (OI) type VII. The mutation introduces cryptic splice sites in intron-3 of CRTAP, resulting in two mature mutant transcripts with cryptic exons. While transcript-1 translates to a truncated isoform (277 amino acids) with thirteen C-terminal non-wild-type amino acids, transcript-2 translates to a wild-type protein sequence, except that this isoform contains an in-frame fusion of non-wild-type twenty-five amino acids in a tetratricopeptide repeat sequence. Both mutant isoforms of CRTAP are unstable due to the presence of a unique 'GWxxI' degron, which finally leads to loss of proline hydroxylation and aggregation of type I collagen. Although type I collagen aggregates undergo autophagy, the overall proteotoxicity resulted in death of the proband cells by senescence. In summary, we present a genetic disease pathomechanism by linking a novel deep intronic mutation in CRTAP to unstable mutant isoforms of the protein in lethal OI type VII.

HYPK coordinates degradation of polyneddylated proteins by autophagy.

Selective degradation of protein aggregates by macroautophagy/autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like proteins, NEDD8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3-interacting region which bind to NEDD8 and LC3 respectively. Both NEDD8 and HYPK are positive modulators of basal and proteotoxicity-induced autophagy, leading to protection of cells from protein aggregates, such as aggregates of mutant HTT exon 1. Thus, we propose an indispensable and additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress.Abbreviations: ATG5, autophagy related 5; ATG12, autophagy related 12; ATG14, autophagy related 14; BECN1, beclin 1; CBL, casitas B-lineage lymphoma; CBLB, Cbl proto-oncogene B; GABARAP, GABA type A receptor-associated protein; GABARAPL1, GABA type A receptor associated protein like 1; GABARAPL2, GABA type A receptor associated protein like 2; GFP, green fluorescent protein; HTT, huntingtin; HTT97Q exon 1, huntingtin 97-glutamine exon 1; HUWE1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; HYPK, huntingtin interacting protein K; IgG, immunoglobulin G; IMR-32, Institute for Medical Research-32; KD, knockdown; Kd, dissociation constant; LAMP1, lysosomal associated membrane protein 1; LIR, LC3 interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAP1LC3A/LC3A, microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MARK1, microtubule affinity regulating kinase 1; MARK2, microtubule affinity regulating kinase 2; MARK3, microtubule affinity regulating kinase 3; MARK4, microtubule affinity regulating kinase 4; MCF7, Michigan Cancer Foundation-7; MTOR, mechanistic target of rapamycin kinase; NAE1, NEDD8 activating enzyme E1 subunit 1; NBR1, NBR1 autophagy cargo receptor; NEDD8, NEDD8 ubiquitin like modifier; Ni-NTA, nickel-nitrilotriacetic acid; NUB1, negative regulator of ubiquitin like proteins 1; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PolyQ, poly-glutamine; PSMD8, proteasome 26S subunit, non-ATPase 8; RAD23A, RAD23 homolog A, nucleotide excision repair protein; RAD23B, RAD23 homolog B, nucleotide excision repair protein; RFP, red fluorescent protein; RPS27A, ribosomal protein S27a; RSC1A1, regulator of solute carriers 1; SNCA, synuclein alpha; SIK1, salt inducible kinase 1; siRNA, small interfering ribonucleic acid; SOD1, superoxide dismutase 1; SPR, surface plasmon resonance; SQSTM1, sequestosome 1; SUMO1, small ubiquitin like modifier 1; TAX1BP1, Tax1 binding protein 1; TDRD3, tudor domain containing 3; TNRC6C, trinucleotide repeat containing adaptor 6C; TOLLIP, toll interacting protein; TUBA, tubulin alpha; TUBB, tubulin beta class I; UBA, ubiquitin-associated; UBA1, ubiquitin like modifier activating enzyme 1; UBA5, ubiquitin like modifier activating enzyme 5; UBAC1, UBA domain containing 1; UBAC2, UBA domain containing 2; UBAP1, ubiquitin associated protein 1; UBAP2, ubiquitin associated protein 2; UBASH3B, ubiquitin associated and SH3 domain containing B; UBD/FAT10, ubiquitin D; UBE2K, ubiquitin conjugating enzyme E2 K; UBLs, ubiquitin-like proteins; UBL7, ubiquitin like 7; UBQLN1, ubiquilin 1; UBQLN2, ubiquilin 2; UBQLN3, ubiquilin 3; UBQLN4, ubiquilin 4; UBXN1, UBX domain protein 1; ULK1, unc-51 like autophagy activating kinase 1; URM1, ubiquitin related modifier 1; USP5, ubiquitin specific peptidase 5; USP13, ubiquitin specific peptidase 13; VPS13D, vacuolar protein sorting 13 homolog D.

The E262K mutation in Lamin A links nuclear proteostasis imbalance to laminopathy-associated premature aging.

Deleterious, mostly de novo, mutations in the lamin A (LMNA) gene cause spatio-functional nuclear abnormalities that result in several laminopathy-associated progeroid conditions. In this study, exome sequencing in a sixteen-year-old male with manifestations of premature aging led to the identification of a mutation, c.784G>A, in LMNA, resulting in a missense protein variant, p.Glu262Lys (E262K), that aggregates in nucleoplasm. While bioinformatic analyses reveal the instability and pathogenicity of LMNAE262K , local unfolding of the mutation-harboring helical region drives the structural collapse of LMNAE262K into aggregates. The E262K mutation also disrupts SUMOylation of lysine residues by preventing UBE2I binding to LMNAE262K , thereby reducing LMNAE262K degradation, aggregated LMNAE262K sequesters nuclear chaperones, proteasomal proteins, and DNA repair proteins. Consequently, aggregates of LMNAE262K disrupt nuclear proteostasis and DNA repair response. Thus, we report a structure-function association of mutant LMNAE262K with toxicity, which is consistent with the concept that loss of nuclear proteostasis causes early aging in laminopathies.

Cellular targets of mefloquine.

Mefloquine is a quinoline-based compound widely used as an antimalarial drug, particularly in chemoprophylaxis. Although decades of research have identified various aspects of mefloquine's anti-Plasmodium properties, toxic effects offset its robust use in humans. Mefloquine exerts harmful effects in several types of human cells by targeting many of the cellular lipids, proteins, and complexes, thereby blocking a number of downstream signaling cascades. In general, mefloquine modulates several cellular phenomena, such as alteration of membrane potential, induction of oxidative stress, imbalance of ion homeostasis, disruption of metabolism, failure of organelle function, etc., leading to cell cycle arrest and programmed cell death. This review aims to summarize the information on functional and mechanistic findings related to the cytotoxic effects of mefloquine.

Differential Stabilities of Mefloquine-Bound Human and Plasmodium falciparum Acyl-CoA-Binding Proteins.

Toxic effects of pharmacological drugs restrict their robust application against human diseases. Although used as a drug in the combinatorial therapy to treat malaria, the use of mefloquine is not highly recommended because of its adverse effects in humans. Mefloquine inhibits the binding of acyl-CoAs to acyl-CoA-binding proteins of Plasmodium falciparum (PfACBPs) and human (hACBP). In this study, we have used molecular dynamics simulation and other computational approaches to investigate the differences of stabilities of mefloquine-PfACBP749 and mefloquine-hACBP complexes. The stability of mefloquine in the binding cavity of PfACBP749 is less than its stability in the binding pocket of hACBP. Although the essential tyrosine residues (tyrosine-30 and tyrosine-33 of PfACBP749 and tyrosine-29 and tyrosine-32 of hACBP) mediate the initial binding of mefloquine to the proteins by π-stacking interactions, additional temporally longer interactions between mefloquine and aspartate-22 and methionine-25 of hACBP result in stronger binding of mefloquine to hACBP. The higher fluctuation of mefloquine-binding residues of PfACBP749 contributes to the instability of mefloquine in the binding cavity of the protein. On the contrary, in the mefloquine-bound state, the stability of hACBP protein is less than the stability of PfACBP749. The helix-to-coil transition of the N-terminal hydrophobic region of hACBP has a destabilizing effect upon the protein's structure. This causes the induction of aggregation properties in the hACBP in the mefloquine-bound state. Taken together, we describe the mechanistic features that affect the differential dynamic stabilities of mefloquine-bound PfACBP749 and hACBP proteins.

The metastable states of proteins.

The intriguing process of protein folding comprises discrete steps that stabilize the protein molecules in different conformations. The metastable state of protein is represented by specific conformational characteristics, which place the protein in a local free energy minimum state of the energy landscape. The native-to-metastable structural transitions are governed by transient or long-lived thermodynamic and kinetic fluctuations of the intrinsic interactions of the protein molecules. Depiction of the structural and functional properties of metastable proteins is not only required to understand the complexity of folding patterns but also to comprehend the mechanisms of anomalous aggregation of different proteins. In this article, we review the properties of metastable proteins in context of their stability and capability of undergoing atypical aggregation in physiological conditions.

Mefloquine binding to human acyl-CoA binding protein leads to redox stress-mediated apoptotic death of human neuroblastoma cells.

Malaria is an infectious disease that is caused by different species of Plasmodium. Several antimalarial drugs are used to counter the spread and infectivity of Plasmodium species. However, humans are also vulnerable to many of the antimalarial drugs, including the quinoline-based drugs. In particular, the antimalarial mefloquine has been reported to show adverse neuropsychiatric effects in humans. Though mefloquine is known to be neurotoxic, the molecular mechanisms associated with this phenomenon are still obscure. In this study, we show that mefloquine binds to and inactivates the human acyl-CoA binding protein (hACBP), potentially inducing redox stress in human neuroblastoma cells (IMR-32). Mefloquine occupies the acyl-CoA binding pocket of hACBP by interacting with several of the critical acyl-CoA binding amino acids. This leads to the competitive inhibition of acyl-CoA(s) binding to hACBP and to the accumulation of lipid droplets inside the IMR-32 cells. The accumulation of cytosolic lipid globules and oxidative stress finally correlates with the apoptotic death of cells. Taken together, our study deciphers a mechanistic detail of how mefloquine leads to the death of human cells by perturbing the activity of hACBP and lipid homeostasis.

T54R mutation destabilizes the dimer of superoxide dismutase 1T54R by inducing steric clashes at the dimer interface.

Mutations cause abnormalities in protein structure, function and oligomerization. Different mutations in the superoxide dismutase 1 (SOD1) protein cause its misfolding, loss of dimerization and aggravate its aggregation in the amyotrophic lateral sclerosis disease. In this study, we report the mechanistic details of how a threonine-to-arginine mutation at the 54th position (T54R) of SOD1 results in destabilization of the dimer interface of SOD1T54R. Using computational and experimental methods, we show that the T54R mutation increases fluctuation of the mutation-harboring loop (R54-loop) of SOD1T54R. Fluctuation of this loop causes steric clashes that involve arginine-54 (R54) and other residues of SOD1T54R, resulting in loss of inter-subunit contacts at the dimer interface. Since the T54 residue-containing loop is necessary for the dimerization of wild-type SOD1, fluctuation of the R54-loop, steric clashes involving R54 and loss of inter-subunit contacts give rise to the loss of SOD1T54R dimer stability. This correlates to energetically unfavorable tethering of the monomers of SOD1T54R. The outcome is gradual splitting of SOD1T54R dimers into monomers, thereby exposing the previously buried hydrophobic interface residues to the aqueous environment. This event finally leads to aggregation of SOD1T54R. T54R mutation has no effect in altering the relative positions of copper and zinc ion binding residues of SOD1T54R. The native SOD1 structure is stable, and there is no destabilizing effect at its dimer interface. Overall, our study reveals the intricate mechanism of T54R mutation-associated destabilization of the dimer of the SOD1T54R protein.