Decoding the molecular mechanism of selective autophagy of glycogen mediated by autophagy receptor STBD1.

Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.

Mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins.

TAX1BP1, a multifunctional autophagy adaptor, plays critical roles in different autophagy processes. As an autophagy receptor, TAX1BP1 can interact with RB1CC1, NAP1, and mammalian ATG8 family proteins to drive selective autophagy for relevant substrates. However, the mechanistic bases underpinning the specific interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins remain elusive. Here, we find that there are two distinct binding sites between TAX1BP1 and RB1CC1. In addition to the previously reported TAX1BP1 SKICH (skeletal muscle and kidney enriched inositol phosphatase (SKIP) carboxyl homology)/RB1CC1 coiled-coil interaction, the first coiled-coil domain of TAX1BP1 can directly bind to the extreme C-terminal coiled-coil and Claw region of RB1CC1. We determine the crystal structure of the TAX1BP1 SKICH/RB1CC1 coiled-coil complex and unravel the detailed binding mechanism of TAX1BP1 SKICH with RB1CC1. Moreover, we demonstrate that RB1CC1 and NAP1 are competitive in binding to the TAX1BP1 SKICH domain, but the presence of NAP1's FIP200-interacting region (FIR) motif can stabilize the ternary TAX1BP1/NAP1/RB1CC1 complex formation. Finally, we elucidate the molecular mechanism governing the selective interactions of TAX1BP1 with ATG8 family members by solving the structure of GABARAP in complex with the non-canonical LIR (LC3-interacting region) motif of TAX1BP1, which unveils a unique binding mode between LIR and ATG8 family protein. Collectively, our findings provide mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins and are valuable for further understanding the working mode and function of TAX1BP1 in autophagy.

Characterization of glomerular basement membrane components within pediatric glomerular diseases.

Background: Disruptions in gene expression associated with the glomerular basement membrane (GBM) could precipitate glomerular dysfunction. Nevertheless, a comprehensive understanding of the characterization of GBM components within pediatric glomerular diseases and their potential association with glomerular function necessitates further systematic investigation. Methods: We conducted a systematic analysis focusing on the pathological transformations and molecular attributes of key constituents within the GBM, specifically Collagen IV α3α4α5, Laminin α5ß2γ1, and Integrin α3ß1, across prevalent pediatric glomerular diseases. Results: We observed upregulation of linear expression levels of COL4A3/4/5 and Laminin 5α proteins, along with a partial reduction in the linear structural expression of Podocin in idiopathic nephrotic syndrome (INS), encompassing minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), but showing a reduction in IgA nephropathy (IgAN), IgA vasculitis nephritis (IgAVN) and lupus nephritis (LN). Furthermore, our study revealed reductions in Laminin ß2γ1 and Integrin α3ß1 in both primary and secondary childhood glomerular diseases. Conclusion: In INS, notably MCD and FSGS, there is a notable increase in the linear expression levels of COL4A3/4/5 and Laminin 5α proteins. In contrast, in IgAN, IgAVN, and LN, there is a consistent reduction in the expression of these markers. Furthermore, the persistent reduction of Laminin ß2γ1 and Integrin α3ß1 in both primary and secondary childhood glomerular diseases suggests a shared characteristic of structural alterations within the GBM across these conditions.

Mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN.

HOIL-1L and SHARPIN are two essential regulatory subunits of the linear ubiquitin chain assembly complex (LUBAC), which is the only known E3 ligase complex generating linear ubiquitin chains. In addition to their LUBAC-dependent functions, HOIL-1L and SHARPIN alone play crucial roles in many LUBAC-independent cellular processes. Importantly, deficiency of HOIL-1L or SHARPIN leads to severe disorders in humans or mice. However, the mechanistic bases underlying the multi-functions of HOIL-1L and SHARPIN are still largely unknown. Here, we uncover that HOIL-1L and SHARPIN alone can form homo-dimers through their LTM motifs. We solve two crystal structures of the dimeric LTM motifs of HOIL-1L and SHARPIN, which not only elucidate the detailed molecular mechanism underpinning the dimer formations of HOIL-1L and SHARPIN, but also reveal a general mode shared by the LTM motifs of HOIL-1L and SHARPIN for forming homo-dimer or hetero-dimer. Furthermore, we elucidate that the polyglucosan body myopathy-associated HOIL-1L A18P mutation disturbs the structural folding of HOIL-1L LTM, and disrupts the dimer formation of HOIL-1L. In summary, our study provides mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN mediated by their LTM motifs, and expands our understandings of the multi-functions of HOIL-1L and SHARPIN as well as the etiology of relevant human disease caused by defective HOIL-1L.

Klotho inhibits renal ox-LDL deposition via IGF-1R/RAC1/OLR1 signaling to ameliorate podocyte injury in diabetic kidney disease.

OBJECTIVE: Diabetic kidney disease (DKD) is characterized by the abnormal deposition of oxidized low-density lipoprotein (ox-LDL), which contributes to podocyte damage. Klotho, an aging suppressor that plays a critical role in protecting podocytes in DKD, is mainly expressed in kidney tubular epithelium and secreted in the blood. However, it has not been established whether Klotho can alleviate podocyte injury by inhibiting renal ox-LDL deposition, and the potential molecular mechanisms require further investigation. METHODS: We conducted a comprehensive analysis of serum and kidney biopsy samples obtained from patients diagnosed with DKD. Additionally, to explore the underlying mechanism of Klotho in the deposition of ox-LDL in the kidneys, we employed a mouse model of DKD with the Klotho genotype induced by streptozotocin (STZ). Furthermore, we conducted meticulous in vitro experiments on podocytes to gain further insights into the specific role of Klotho in the deposition of ox-LDL within the kidney. RESULTS: Our groundbreaking study unveiled the remarkable ability of the soluble form of Klotho to effectively inhibit high glucose-induced ox-LDL deposition in podocytes affected by DKD. Subsequent investigations elucidated that Klotho achieved this inhibition by reducing the expression of the insulin/insulin-like growth factor 1 receptor (IGF-1R), consequently leading to a decrease in the expression of Ras-related C3 botulinum toxin substrate 1 (RAC1) and an enhancement of mitochondrial function. Ultimately, this series of events culminated in a significant reduction in the expression of the oxidized low-density lipoprotein receptor (OLR1), thereby resulting in a notable decrease in renal ox-LDL deposition in DKD. CONCLUSION: Our findings suggested that Klotho had the potential to mitigate podocyte injury and reduced high glucose-induced ox-LDL deposition in glomerulus by modulating the IGF-1R/RAC1/OLR1 signaling. These results provided valuable insights that could inform the development of novel strategies for diagnosing and treating DKD.

Mechanistic insights into the enzymatic activity of E3 ligase HOIL-1L and its regulation by the linear ubiquitin chain binding.

Heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) serves as a unique E3 ligase to catalyze the mono-ubiquitination of relevant protein or sugar substrates and plays vital roles in numerous cellular processes in mammals. However, the molecular mechanism underpinning the E3 activity of HOIL-1L and the related regulatory mechanism remain elusive. Here, we report the crystal structure of the catalytic core region of HOIL-1L and unveil the key catalytic triad residues of HOIL-1L. Moreover, we discover that HOIL-1L contains two distinct linear di-ubiquitin binding sites that can synergistically bind to linear tetra-ubiquitin, and the binding of HOIL-1L with linear tetra-ubiquitin can promote its E3 activity. The determined HOIL-1L/linear tetra-ubiquitin complex structure not only elucidates the detailed binding mechanism of HOIL-1L with linear tetra-ubiquitin but also uncovers a unique allosteric ubiquitin-binding site for the activation of HOIL-1L. In all, our findings provide mechanistic insights into the E3 activity of HOIL-1L and its regulation by the linear ubiquitin chain binding.

A high-impact FN1 variant correlates with fibronectin-mediated glomerulopathy via decreased binding to collagen type IV.

The glomerular basement membrane (GBM) consists of laminins, collagen IV, nidogens, and fibronectin and is essential for filtration barrier integrity in the kidney. Critically, structural and functional abnormalities in the GBM are involved in chronic kidney disease (CKD) occurrence and development. Fibronectin is encoded by FN1 and is essential for podocyte-podocyte and podocyte-matrix interactions. However, disrupted or disordered fibronectin occurs in many kidney diseases. In this study, we identified a novel mutation (c.3415G>A) in FN1 that causes glomerular fibronectin-specific deposition in a gain-of-function manner, that may be associated with thin basement membrane nephropathy (TBMN) and expand the spectrum of phenotypes seen in glomerulopathy with fibronectin deposits (GFND). Our studies confirmed this variant increased fibronectin's ability to bind to integrin, thereby maintaining podocyte adhesion. Also, we hypothesised that TBMN arose as the fibronectin variant exhibited a decreased capacity to bind COL4A3/4. Our study is the first to identify and link this novel pathogenic mutation (c.3415G>A) in FN1 to GFND as well as TBMN, which may broaden the phenotype and mutation spectrums of the FN1 gene. We believe our data will positively impact genetic counselling and prenatal diagnostics for GFND with TBMN and other associated conditions that may be commonly benign conditions in humans, and may not require proteinuria-lowering treatments or renal biopsy.

Novel pathogenic variants in CUBN uncouple proteinuria from renal function.

BACKGROUND: Proteinuria is an unfavorable clinical condition highly associated with a risk of renal and cardiovascular disease in chronic kidney disease (CKD). However, whether all proteinuria forms are linked to renal impairment are still unclear. Cubilin is an endocytic receptor highly expressed in renal proximal tubules mediating uptake of albumin, transferrin and α1-microglobulin. METHODS: Exome sequencing method initially identified candidate genes. With the application of exome sequencing combined with Sanger sequencing, we further focused on CUBN through bioinformatics analysis. The pathogenic effects of the potentially causative variants were verified utilizing complementary analysis of clinical data and systematic characterization of the variants' expression and function with clinical samples and in vitro experiments in HEK293T cell lines along with in vivo experiments in mice. RESULTS: In this study, we identified four novel variants locating after the vitamin B12 (vitB12)-binding domain of Cubilin (encoded by CUBN, NM_001081.3: c.4397G > A (p.C1466Y), c.6796C > T (p.R2266X), c.6821 + 3A > G and c.5153_5154delCT (p.S1718X)) in two families. Moreover, the variants severely affected the expression and function of Cubilin in renal proximal tubules and caused albuminuria, increasing levels in urine transferrin and α1-microglobulin, but without progressive glomerular filtration barrier (GFB) impairment, vitB12 deficiencies or abnormal blood levels of HDL and albumin. Further mechanistic insights showed that the variants after the vitB12-binding domain of CUBN merely disrupted the association with Amnionless (AMN) that exhibited aberrant localization in cell cytoplasm rather than membrane. CONCLUSIONS: Here, our findings suggested that different mutation types after the vitB12-binding domain of CUBN uncouple proteinuria from glomerular filtration barrier, that may be an unexpectedly common benign condition in humans and may not require any proteinuria-lowering treatment or renal biopsy.

Structure of the receptor-activated human TRPC6 and TRPC3 ion channels.

TRPC6 and TRPC3 are receptor-activated nonselective cation channels that belong to the family of canonical transient receptor potential (TRPC) channels. They are activated by diacylglycerol, a lipid second messenger. TRPC6 and TRPC3 are involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high-affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 Å resolution. We also present the structure of human TRPC3 at 4.4 Å resolution. These structures show two-layer architectures in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domains, including the N-terminal ankyrin repeats and the C-terminal coiled-coil, contribute to the tetramer assembly. The high-affinity inhibitor BTDM wedges between the S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structures uncover the molecular architecture of TRPC channels and provide a structural basis for understanding the mechanism of these channels.

Activation of peripheral KCNQ channels relieves gout pain.

Intense inflammatory pain caused by urate crystals in joints and other tissues is a major symptom of gout. Among therapy drugs that lower urate, benzbromarone (BBR), an inhibitor of urate transporters, is widely used because it is well tolerated and highly effective. We demonstrate that BBR is also an activator of voltage-gated KCNQ potassium channels. In cultured recombinant cells, BBR exhibited significant potentiation effects on KCNQ channels comparable to previously reported classical activators. In native dorsal root ganglion neurons, BBR effectively overcame the suppression of KCNQ currents, and the resultant neuronal hyperexcitability caused by inflammatory mediators, such as bradykinin (BK). Benzbromarone consistently attenuates BK-, formalin-, or monosodium urate-induced inflammatory pain in rat and mouse models. Notably, the analgesic effects of BBR are largely mediated through peripheral and not through central KCNQ channels, an observation supported both by pharmacokinetic studies and in vivo experiments. Moreover, multiple residues in the superficial part of the voltage sensing domain of KCNQ channels were identified critical for the potentiation activity of BBR by a molecular determinant investigation. Our data indicate that activation of peripheral KCNQ channels mediates the pain relief effects of BBR, potentially providing a new strategy for the development of more effective therapies for gout.