Site-specific photo-crosslinking of Hsc70 with the KFERQ pentapeptide motif in a chaperone-mediated autophagy and microautophagy substrate in mammalian cells.

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.

Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

1H, 13C, and 15N resonance assignments and solution structure of the N-terminal divergent calponin homology (NN-CH) domain of human intraflagellar transport protein 54.

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.

Serum anti­KIAA0513 antibody as a common biomarker for mortal atherosclerotic and cancerous diseases.

Numerous antibody biomarkers have been reported for cancer and atherosclerosis-related diseases. The major complications of atherosclerosis and diabetes mellitus (DM) are acute ischemic stroke (AIS), cardiovascular disease (CVD) and chronic kidney disease (CKD). Cancer development is accompanied by arterial disorders, such as angiogenesis and atherosclerosis, and DM is a risk factor for the development of certain types of cancer. Atherosclerosis-related diseases and cancers are therefore interrelated and could be detected using a common biomarker. In the present study, the initial screening using the protein array method identified KIAA0513 as an antigen recognized by serum IgG antibodies in patients with atherosclerosis. The amplified luminescent proximity homogeneous assay-linked immunosorbent assay revealed significantly higher serum antibody levels against recombinant KIAA0513 protein in patients with AIS, transient ischemic attack (TIA), DM, CVD, obstructive sleep apnea syndrome (OSAS), CKD and solid cancers, such as esophageal, gastric, colon, lung and breast cancers, compared with healthy donors. A receiver operating characteristic (ROC) analysis revealed that the highest areas under the ROC curves of anti-KIAA0513 antibodies were obtained for esophageal cancer, nephrosclerosis-type CKD and DM. Spearman's correlation analysis revealed that serum anti-KIAA0513 antibody levels were associated with maximum intima-media thickness and plaque score, which are indices of atherosclerosis and stenosis. Serum anti-KIAA0513 antibody markers appear to be useful for diagnosing AIS, TIA, DM, CVD, OSAS, CKD and solid cancers, and may reflect common arterial alterations leading to atherosclerotic and cancerous diseases.

A Potent PDK4 Inhibitor for Treatment of Heart Failure with Reduced Ejection Fraction.

Heart failure with reduced ejection fraction (HFrEF) is characterized not only by reduced left ventricular ejection fraction (EF) but is also combined with symptoms such as dyspnea, fatigue, and edema. Several pharmacological interventions have been established. However, a treatment targeting a novel pathophysiological mechanism is still needed. Evidence indicating that inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may be cardioprotective has been accumulating. Thus, we focused on vitamin K3 and used its framework as a new PDK4 inhibitor skeleton to synthesize new PDK4 inhibitors that show higher activity than the existing PDK4 inhibitor, dichloroacetic acid, and tested their cardioprotective effects on a mouse heart failure model. Among these inhibitors, PDK4 inhibitor 8 improved EF the most, even though it did not reverse cardiac fibrosis or wall thickness. This novel, potent PDK4 inhibitor may improve EF of failing hearts by regulating bioenergetics via activation of the tricarboxylic acid cycle.