Voltage-gated K+ (KV) channels govern K+ ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our findings demonstrate that KV cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types.

Latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer.

Histone lysine demethylases (KDMs) play an essential role in biological processes such as transcription regulation, RNA maturation, transposable element control, and genome damage sensing and repair. In most cases, their action requires catalytic activities, but non-catalytic functions have also been shown in some KDMs. Indeed, some strictly KDM-related proteins and some KDM isoforms do not act as histone demethylase but show other enzymatic activities or relevant non-enzymatic functions in different cell types. Moreover, many studies have reported on functions potentially supported by catalytically dead mutant KDMs. This is probably due to the versatility of the catalytical core, which can adapt to assume different molecular functions, and to the complex multi-domain structure of these proteins which encompasses functional modules for targeting histone modifications, promoting protein-protein interactions, or recognizing nucleic acid structural motifs. This rich modularity and the availability of multiple isoforms in the various classes produced variants with enzymatic functions aside from histone demethylation or variants with non-catalytical functions during the evolution. In this review we will catalog the proteins with null or questionable demethylase activity and predicted or validated inactive isoforms, summarizing what is known about their alternative functions. We will then go through some experimental evidence for the non-catalytical functions of active KDMs.

The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C-terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex.

Actin, which plays a crucial role in cellular structure and function, interacts with various binding proteins, notably myosin. In mammals, actin is composed of six isoforms that exhibit high levels of sequence conservation and structural similarity overall. As a result, the selection of actin isoforms was considered unimportant in structural studies of their binding with myosin. However, recent high-resolution structural research discovered subtle structural differences in the N-terminus of actin isoforms, suggesting the possibility that each actin isoform may engage in specific interactions with myosin isoforms. In this study, we aimed to explore this possibility, particularly by understanding the influence of different actin isoforms on the interaction with myosin 7A. First, we compared the reported actomyosin structures utilizing the same type of actin isoforms as the high-resolution filamentous skeletal α-actin (3.5 Å) structure elucidated using cryo-EM. Through this comparison, we confirmed that the diversity of myosin isoforms leads to differences in interaction with the actin N-terminus, and that loop 2 of the myosin actin-binding sites directly interacts with the actin N-terminus. Subsequently, with the aid of multiple sequence alignment, we observed significant variations in the length of loop 2 across different myosin isoforms. We predicted that these length differences in loop 2 would likely result in structural variations that would affect the interaction with the actin N-terminus. For myosin 7A, loop 2 was found to be very short, and protein complex predictions using skeletal α-actin confirmed an interaction between loop 2 and the actin N-terminus. The prediction indicated that the positively charged residues present in loop 2 electrostatically interact with the acidic patch residues D24 and D25 of actin subdomain 1, whereas interaction with the actin N-terminus beyond this was not observed. Additionally, analyses of the actomyosin-7A prediction models generated using various actin isoforms consistently yielded the same results regardless of the type of actin isoform employed. The results of this study suggest that the subtle structural differences in the N-terminus of actin isoforms are unlikely to influence the binding structure with short loop 2 myosin 7A. Our findings are expected to provide a deeper understanding for future high-resolution structural binding studies of actin and myosin.

Proteomics, the study of proteins within biological systems, has seen remarkable advancements in recent years, with protein isoform detection emerging as one of the next major frontiers. One of the primary challenges is achieving the necessary peptide and protein coverage to confidently differentiate isoforms as a result of the protein inference problem and protein false discovery rate estimation challenge in large data. In this chapter, we describe the application of artificial intelligence-assisted peptide property prediction for database search engine rescoring by Oktoberfest, an approach that has proven effective, particularly for complex samples and extensive search spaces, which can greatly increase peptide coverage. Further, it illustrates a method for increasing isoform coverage by the PickedGroupFDR approach that is designed to excel when applied on large data. Real-world examples are provided to illustrate the utility of the tools in the context of rescoring, protein grouping, and false discovery rate estimation. By implementing these cutting-edge techniques, researchers can achieve a substantial increase in both peptide and isoform coverage, thus unlocking the potential of protein isoform detection in their studies and shedding light on their roles and functions in biological processes.

Proteogenomics has revealed the translation of unannotated open reading frames (ORFs) present in mRNAs and in noncoding RNAs (ncRNAs). OpenProt annotates all ORFs with a minimum of 30 codons in the transcriptome of several species and displays many functional features associated with the corresponding proteins. Two types of proteins are annotated: reference or canonical proteins which are proteins already annotated in UniProt, RefSeq, or Ensembl and noncanonical proteins. Noncanonical proteins form two groups: predicted novel isoforms that display a significant level of homology with a reference protein and alternative proteins that are new proteins with no significant homology to known proteins. This chapter describes how to check whether a gene and/or transcript contains multiple open reading frames and how to use OpenProt databases for the detection of alternative proteins and novel isoforms by mass spectrometry-based proteomics.

The fly Drosophila yakuba has lost an ancestral component of the male courtship song: this is due to ontogenetic death of effector neurons in the ventral nerve cord, a result of the D. yakuba sex-determining gene dsx producing a male isoform, dsxM, with cell-death-promoting activity similar to that of the female isoform, dsxF, in D. melanogaster.

This study investigates various pathological tau isoforms in the retina of individuals with early and advanced Alzheimer\'s disease (AD), exploring their connection with disease status. Retinal cross-sections from predefined superior-temporal and inferior-temporal subregions and corresponding brains from neuropathologically confirmed AD patients with a clinical diagnosis of either mild cognitive impairment (MCI) or dementia (n = 45) were compared with retinas from age- and sex-matched individuals with normal cognition (n = 30) and non-AD dementia (n = 4). Retinal tau isoforms, including tau tangles, paired helical filament of tau (PHF-tau), oligomeric-tau (Oligo-tau), hyperphosphorylated-tau (p-tau), and citrullinated-tau (Cit-tau), were stereologically analyzed by immunohistochemistry and Nanostring GeoMx digital spatial profiling, and correlated with clinical and neuropathological outcomes. Our data indicated significant increases in various AD-related pretangle tau isoforms, especially p-tau (AT8, 2.9-fold, pS396-tau, 2.6-fold), Cit-tau at arginine residue 209 (CitR209-tau; 4.1-fold), and Oligo-tau (T22+, 9.2-fold), as well as pretangle and mature tau tangle forms like MC-1-positive (1.8-fold) and PHF-tau (2.3-fold), in AD compared to control retinas. MCI retinas also exhibited substantial increases in Oligo-tau (5.2-fold), CitR209-tau (3.5-fold), and pS396-tau (2.2-fold). Nanostring GeoMx analysis confirmed elevated retinal p-tau at epitopes: Ser214 (2.3-fold), Ser396 (2.6-fold), Ser404 (2.4-fold), and Thr231 (1.8-fold), particularly in MCI patients. Strong associations were found between retinal tau isoforms versus brain pathology and cognitive status: a) retinal Oligo-tau vs. Braak stage, neurofibrillary tangles (NFTs), and CDR cognitive scores (ρ = 0.63-0.71), b) retinal PHF-tau vs. neuropil threads (NTs) and ABC scores (ρ = 0.69-0.71), and c) retinal pS396-tau vs. NTs, NFTs, and ABC scores (ρ = 0.67-0.74). Notably, retinal Oligo-tau strongly correlated with retinal Aβ42 and arterial Aβ40 forms (r = 0.76-0.86). Overall, this study identifies and quantifies diverse retinal tau isoforms in MCI and AD patients, underscoring their link to brain pathology and cognition. These findings advocate for further exploration of retinal tauopathy biomarkers to facilitate AD detection and monitoring via noninvasive retinal imaging.

In the developing human brain, only 53 stochastically expressed clustered protocadherin (cPcdh) isoforms enable neurites from individual neurons to recognize and self-avoid while simultaneously maintaining contact with neurites from other neurons. Cell assays have demonstrated that self-recognition occurs only when all cPcdh isoforms perfectly match across the cell boundary, with a single mismatch in the cPcdh expression profile interfering with recognition. It remains unclear, however, how a single mismatched isoform between neighboring cells is sufficient to block erroneous recognitions. Using systematic cell aggregation experiments, we show that abolishing cPcdh interactions on the same membrane (cis) results in a complete loss of specific combinatorial binding between cells (trans). Our computer simulations demonstrate that the organization of cPcdh in linear array oligomers, composed of cis and trans interactions, enhances self-recognition by increasing the concentration and stability of cPcdh trans complexes between the homotypic membranes. Importantly, we show that the presence of mismatched isoforms between cells drastically diminishes the concentration and stability of the trans complexes. Overall, we provide an explanation for the role of the cPcdh assembly arrangements in neuronal self/non-self-discrimination underlying neuronal self-avoidance.