BACKGROUND: Familial hypercholesterolemia (FH) is a common inherited metabolic disease that causes premature atherosclerosis, cardiovascular disease, and even death at a young age. Approximately 95% of FH-causing genetic variants that have been identified are in the LDLR gene. However, only 10% of the FH population worldwide has been diagnosed and adequately treated, due to the existence of numerous unidentified variants, uncertainties in the pathogenicity scoring of many variants, and a substantial number of individuals lacking access to genetic testing.
OBJECTIVE: The aim of this study was to identify a novel variant in the LDLR gene that causes FH in a Chinese family, thereby expanding the spectrum of FH-causing variants.
METHODS: Patients were recruited from Beijing Anzhen Hospital, Capital Medical University. FH diagnosis was made according to the Dutch Lipid Clinical Network (DLCN) criteria. Whole-exome sequencing (WES) was conducted to identify the FH-causing variant in the proband, and amplicon sequencing was used to verify the variant in his family members.
RESULTS: A three-generation Chinese family was recruited, and two FH patients were clinically diagnosed, both without known FH-causing variants. These two FH patients and another possible patient carried a novel variant, NC_000019.9(NM_000527.5):c.89_92dup (NP_000518.1:p.Phe32Argfs*21), in the ligand-binding domain of the low-density lipoprotein (LDL) receptor that led to a frameshift. The FH adults in the family showed severe clinical symptoms and statin therapy resistance.
CONCLUSIONS: This study identified a novel pathogenic LDLR variant, c.89_92dup, associated with severe FH clinical manifestations and statin therapy resistance.

Mutations in the androgen receptor (AR) ligand-binding domain (LBD) can cause resistance to drugs used to treat prostate cancer. Commonly found mutations include L702H, W742C, H875Y, F877L and T878A, while the F877L mutation can convert second-generation antagonists such as enzalutamide and apalutamide into agonists. However, pruxelutamide, another second-generation AR antagonist, has no agonist activity with the F877L and F877L/T878A mutants and instead maintains its inhibitory activity against them. Here, it is shown that the quadruple mutation L702H/H875Y/F877L/T878A increases the soluble expression of AR LBD in complex with pruxelutamide in Escherichia coli. The crystal structure of the quadruple mutant in complex with the agonist dihydrotestosterone (DHT) reveals a partially open conformation of the AR LBD due to conformational changes in the loop connecting helices H11 and H12 (the H11-H12 loop) and Leu881. This partially open conformation creates a larger ligand-binding site for AR. Additional structural studies suggest that both the L702H and F877L mutations are important for conformational changes. This structural variability in the AR LBD could affect ligand binding as well as the resistance to antagonists.

RORγt plays an important role in mediating IL-17 production and some tumor cells. It has four functional domains, of which the ligand-binding domain (LBD) is responsible for binding agonists to recruit co-activators or inverse agonists to prevent co-activator recruiting the agonists. Thus, potent ligands targeting the LBD of this protein could provide novel treatments for cancer and autoimmune diseases. In this perspective, we summarized and discussed various modes of action (MOA) of RORγt-ligand binding structures. The ligands can bind with RORγt at either orthosteric site or the allosteric site, and the binding modes at these two sites are different for agonists and inverse agonist. At the orthosteric site, the binding of agonist is to stabilize the H479-Y502-F506 triplet interaction network of RORγt. The binding of inverse agonist features as these four apparent ways: (1) blocking the entrance of the agonist pocket in RORγt; (2) directly breaking the H479-Y502 pair interactions; (3) destabilizing the triplet H479-Y502-F506 interaction network through perturbing the conformation of the side chain in M358 at the bottom of the binding pocket; (4) and destabilizing the triplet H479-Y502-F506 through changing the conformation of the side chain of residue W317 side chain. At the allosteric site of RORγt, the binding of inverse agonist was found recently to inhibit the activation of protein by interacting directly with H12, which results in unfolding of helix 11\' and orientation of H12 to directly block cofactor peptide binding. This overview of recent advances in the RORγt structures is expected to provide a guidance of designing more potent drugs to treat RORγt-related diseases.

Retinoid X receptor (RXRα) is a nuclear receptor (NR) for retinoic acid (RA) and regulates various NR signaling pathways. Ligand-binding domain (LBD) of RXRα can bind with its ligand 9-cis-RA and cofactors, and mediate the forming of homodimer and homotetramer of RXRα and its heterodimer with other NRs, conferring RXRα the ability to play complicated roles in development and diseases. Due to the coexistence of monomer, dimer and tetramer, there are difficulties to study the structure and interaction of RXRα-LBD with its ligands and cofactors in solution and to distinguish the roles of different forms of RXRα in cell. Here, through analyzing available structures of RXRα-LBD, we selected two residues, D379 and L420, in the homodimer interface to design three mutants of RXRα-LBD. Recombinant proteins of the three mutants showed decreased proportions of dimer and tetramer but unchanged overall structure and binding affinities to 9-cis-RA, corepressor SMRT, and coactivator SRC2. Especially, the double-site mutant RXRα-LBDD379A-L420G existed as a uniform monomer. Furthermore, L420 was found to play a similar role in forming RXRα-LBD homodimer and its heterodimer with various NRs, while the role of D379 varies a lot, as it shows almost no interaction with RARα/β, LXRα/β, and THRα/β. This study provides a new insight into the mechanism for forming RXRα-LBD homodimer and its heterodimer with other NRs, and will facilitate the studies on the structure and interaction of RXRα-LBD with ligands, cofactors and drugs in solution, and the broad physiological functions of RXRα cooperating with various NRs in cell.

BACKGROUND: Androgen insensitivity syndrome is an X-linked recessive genetic disease caused by mutations in the androgen receptor gene (AR). However, the underlying molecular mechanisms for the majority of AR variants remain unclear. In this study, we identified a point variant in three patients with complete androgen insensitivity syndrome (CAIS), summarized the correlation analysis, and performed a literature review.
METHODS: The proband was raised as a girl. In infancy, she was first referred to hospital with a right inguinal hernia. Ultrasonography revealed the absence of a uterus and ovaries, and a testis-like structure located at the inguinal canal. Further diagnostic workup detected a 46, XY karyotype, and fluorescence in situ hybridization analysis showed the presence of the SRY gene. Histological analysis revealed the excised tissue to be testicular. Twelve years later, she was admitted to our hospital with a lack of breast development. Her pubic hair and breasts were Tanner stage I. She had normal female external genitalia. Blood hormone tests showed normal testosterone levels, low estradiol levels, and high gonadotropin levels. Her two siblings underwent similar examinations, and all three had a rare hemizygous missense mutation in AR: c.2678C>T. In vitro functional analyses revealed decreased nuclear translocation in AR-c.2678C>T mutation cells.
CONCLUSIONS: This case of CAIS was caused by an AR variant (c.2678C>T). Functional studies showed impaired nuclear translocation ability of the mutant protein.

The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates a wide range of biological and toxicological effects by binding to specific ligands. AhR ligands exist in various internal and external ecological systems, such as in a wide variety of hydrophobic environmental contaminants and naturally occurring chemicals. Most of these ligands have shown differential responses among different species. Understanding the differences and their mechanisms helps in designing better experimental animal models, improves our understanding of the environmental toxicants related to AhR, and helps to screen and develop new drugs. This review systematically discusses the species differences in AhR activation effects and their modes of action. We focus on the species differences following AhR activation from two aspects: (1) the molecular configuration and activation of AhR and (2) the contrast of cis-acting elements corresponding to AhR. The variations in the responses seen in humans and other species following the activation of the AhR signaling pathway can be attributed to both factors.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2\'s inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

Insulin-like growth factor-binding proteins (IGFBPs) are multifunctional proteins that possess IGF-dependent and -independent actions. Recent studies suggest that its IGF-independent action appeared early and that the IGF-binding function may have been acquired later in evolution. The timing of the emergence of IGF-dependent actions is unclear. Here, we identified and characterized an igfbp gene from sea lamprey, an agnathan, which was separated from the jawed vertebrates 450 million years ago. Phylogenetic and structural analyses suggested that the encoded protein belongs to the IGFBP-3 clade in the IGFBP family. Lamprey IGFBP-3 contains an IGF-binding domain (IBD), nuclear localization signal, and transactivation (TA) domain. Biochemical and functional analyses showed that these domains are all functional. Lamprey IGFBP-3 can bind IGFs and modulate IGF signaling when tested in mammalian cells. Lamprey IGFBP-3 also has the capacity to enter the nucleus and has strong TA activity. Forced expression of lamprey IGFBP-3, but not its IBD mutant, in zebrafish embryos decreased body growth and developmental speed. Lamprey IGFBP-3 inhibited BMP2 signaling in cultured cells and in zebrafish embryos, and this action is independent of its IGF-binding function. These results suggest that lamprey IGFBP-3 has both IGF-dependent and -independent actions and provide new insights into the functional evolution of the IGFBP family.

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.