Magdalena Preciado López

One of the main hallmarks of aging is aging-associated inflammation, also known as inflammaging. In this study, by comparing plasma and kidney proteome profiling of young and old mice using LC–MS profiling, we discovered that immunoglobulins are the proteins that exhibit the highest increase with age. This observation seems to have been disregarded because conventional proteome profiling experiments typically overlook the expression of high-abundance proteins or employ depletion methods to remove them before LC–MS analysis. We show that proteome profiling of immunoglobulins will likely be a useful biomarker of aging. Spatial profiling using immunofluorescence staining of kidney sections indicates that the main increases in immunoglobulins with age are localized in the glomeruli of the kidney. Using laser capture microdissection coupled with LC–MS, we show an increase in multiple immune-related proteins in glomeruli from aged mice. Increased deposition of immunoglobulins, immune complexes, and complement proteins in the kidney glomeruli may be a factor leading to reduced filtering capacity of the kidney with age. Therapeutic strategies to reduce the deposition of immunoglobulins in the kidney may be an attractive strategy for healthy aging.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein alpha-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced alpha-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble alpha-Klotho in biofluids. Here, we describe the discovery of peptides that recognize alpha-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to alpha-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich alpha-Klotho from cell lysates and to label alpha-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions to these topologies by PPI-focused therapeutics and advance their development, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand. Cysteine mutants of the ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a system with the 9-mer peptide antigen VL9 and MHC class I receptor HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 hours. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94─NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

α‐Klotho, an aging‐related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co‐receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α‐Klotho are a hallmark of age‐associated diseases. Detecting or labeling α‐Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single‐shot parallel automated fast‐flow synthesis that recognize α‐Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α‐Klotho in physiological settings.

α-Klotho is a protein associated with aging that is expressed in the kidney, parathyroid gland, and choroid plexus. As a transmembrane protein, it acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. α-Klotho has an extracellular domain that can be cleaved, released and circulated in the blood stream as a soluble form. Decreased levels of α-Klotho are an indication of chronic kidney disease and other age-associated diseases. Detecting or labeling transmembrane and soluble α-Klotho is a longstanding challenge that has impeded the in-depth understanding of its role. Here we describe branched multimeric peptides that recognize α-Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched α-Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells. Our results demonstrate the potential of automated flow technology to deliver peptide-based reagents with complex architecture and improved affinity for the selective binding of target proteins in physiological settings.