Orlacchio, A. | Search bioRxiv

bioRxiv2025

Targeted plasma proteomics uncover novel proteins associated with KIF5A-linked SPG10 and ALS spectrum disorders

KIF5A (Kinesin family member 5A) is a motor protein that functions as a key component of the axonal transport machinery. Variants in KIF5A are linked to several neurodegenerative diseases, mainly spastic paraplegia type 10 (SPG10), Charcot-Marie-Tooth disease type 2 (CMT2), and amyotrophic lateral sclerosis (ALS). These diseases share motor neuron involvement but vary significantly in clinical presentation, severity, and progression. KIF5A variants are mainly categorized into N-terminal variants associated with SPG10/CMT2 and C-terminal variants linked to ALS. This study utilized a novel multiplex NULISA targeted platform to analyze plasma proteome from KIF5A-linked SPG10, ALS patients and compared to healthy controls. Our results revealed distinct proteomic signatures, with significant alterations in proteins related to synaptic function, and inflammation. Notably, neurofilament light polypeptide, a biomarker for neurodegenerative diseases, was elevated in KIF5A ALS but not in SPG10 patients. Moreover, these findings can now be taken forward to gain mechanistic understanding of axonopathies linking to N-vs C-terminal KIF5A variants affecting both central and peripheral nervous systems.

neuroscience↗

bioRxiv2024

An in vivo 'turning model' reveals new RanBP9 interactions in lung macrophages.

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.

cell biology↗

bioRxiv2020

IWS1 phosphorylation by AKT kinase controls the nucleocytoplasmic export of type I IFNs and the sensitivity of lung adenocarcinoma cells to oncolytic viral infection, through U2AF2 RNA splicing.

The authors have withdrawn their manuscript. While attempting to reproduce the data on the alternative splicing of exon 2 of U2AF2, they observed that the proposed splicing mechanism could not give rise to a functional U2AF2 protein. In addition, they observed evidence of manipulation in the electropherogram of the splicing junction between exons 1 and 3 and in the primary data on which this electropherogram was based, which were deposited in Mendeley by the first author. These observations raise questions on the integrity of the reported results. In light of this information, the authors have no confidence in the key findings of the paper, and therefore, do not wish it to be cited. If you have any questions, please contact the corresponding author.

cancer biology↗

bioRxiv2020

IWS1 phosphorylation promotes cell proliferation and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the cell cycle-regulated U2AF2 RNA splicing.

The authors have withdrawn their manuscript. While attempting to reproduce the data on the alternative splicing of exon 2 of U2AF2, they observed that the proposed splicing mechanism could not give rise to a functional U2AF2 protein. In addition, they observed evidence of manipulation in the electropherogram of the splicing junction between exons 1 and 3 and in the primary data on which this electropherogram was based, which were deposited in Mendeley by the first author. These observations raise questions on the integrity of the reported results. In light of this information, the authors have no confidence in the key findings of the paper, and therefore, do not wish it to be cited. If you have any questions, please contact the corresponding author.

cancer biology↗

bioRxiv2019

Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo.

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a novel interaction with Nucleolin, a prominent nucleolar protein. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available tag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially reveal novel functions of this protein in vivo such as those linked to Nucleolin.

bioengineering↗