SPRTN is involved in hepatocellular carcinoma development through the ER stress response.

Endoplasmic reticulum (ER) stress, prompted by the accumulation of misfolded or unfolded proteins, triggers the activation of the unfolded protein response (UPR) pathway to restore ER homeostasis. This stress response is implicated in the development of hepatocellular carcinoma (HCC). A biallelic mutation in SPRTN is currently the only known single-gene mutation implicated in the early onset of HCC. However, the exact mechanism linking SPRTN mutations to HCC remains unclear. In our study, we analyzed SPRTN and UPR in 21 human HCC tissue samples using RT-qPCR, immunoblot, and immunohistochemistry. We found alterations in the expression levels of SPRTN and UPR-related genes and proteins in HCC samples. The impact of SPRTN on the ER stress response was assessed in SPRTN-depleted HepG2 cells through RNA sequencing, pull-down assay, comet assay, and mitotic index calculation. We demonstrated that SPRTN interacts with the UPR sensor GRP78. Furthermore, we observed a decrease in SPRTN levels during ER stress, and increased sensitivity to ER stress in SPRTN-depleted cells. These findings suggest an essential role for SPRTN in the ER stress response and provide new insights into HCC pathogenesis. This newly discovered function of SPRTN could significantly enhance our understanding and treatment of HCC.

Co-Aggregation and Parallel Aggregation of Specific Proteins in Major Mental Illness.

BACKGROUND: Disrupted proteostasis is an emerging area of research into major depressive disorder. Several proteins have been implicated as forming aggregates specifically in the brains of subsets of patients with psychiatric illnesses. These proteins include CRMP1, DISC1, NPAS3 and TRIOBP-1. It is unclear, however, whether these proteins normally aggregate together in the same individuals and, if so, whether each protein aggregates independently of each other ("parallel aggregation") or if the proteins physically interact and aggregate together ("co-aggregation"). MATERIALS AND METHODS: Post mortem insular cortex samples from major depressive disorder and Alzheimer's disease patients, suicide victims and control individuals had their insoluble fractions isolated and tested by Western blotting to determine which of these proteins are insoluble and, therefore, likely to be aggregating. The ability of the proteins to co-aggregate (directly interact and form common aggregate structures) was tested by systematic pairwise expression of the proteins in SH-SY5Y neuroblastoma cells, which were then examined by immunofluorescent microscopy. RESULTS: Many individuals displayed multiple insoluble proteins in the brain, although not enough to imply interaction between the proteins. Cell culture analysis revealed that only a few of the proteins analyzed can consistently co-aggregate with each other: DISC1 with each of CRMP1 and TRIOBP-1. DISC1 was able to induce aggregation of full length TRIOBP-1, but not individual domains of TRIOBP-1 when they were expressed individually. CONCLUSIONS: While specific proteins are capable of co-aggregating, and appear to do so in the brains of individuals with mental illness and potentially also with suicidal tendency, it is more common for such proteins to aggregate in a parallel manner, through independent mechanisms. This information aids in understanding the distribution of protein aggregates among mental illness patients and is therefore important for any future diagnostic or therapeutic approaches based on this aspect of mental illness pathology.

TRIOBP-1 Protein Aggregation Exists in Both Major Depressive Disorder and Schizophrenia, and Can Occur through Two Distinct Regions of the Protein.

The presence of proteinopathy, the accumulation of specific proteins as aggregates in neurons, is an emerging aspect of the pathology of schizophrenia and other major mental illnesses. Among the initial proteins implicated in forming such aggregates in these conditions is Trio and F-actin Binding Protein isoform 1 (TRIOBP-1), a ubiquitously expressed protein involved in the stabilization of the actin cytoskeleton. Here we investigate the insolubility of TRIOBP-1, as an indicator of aggregation, in brain samples from 25 schizophrenia patients, 25 major depressive disorder patients and 50 control individuals (anterior cingulate cortex, BA23). Strikingly, insoluble TRIOBP-1 is considerably more prevalent in both of these conditions than in controls, further implicating TRIOBP-1 aggregation in schizophrenia and indicating a role in major depressive disorder. These results were only seen using a high stringency insolubility assay (previously used to study DISC1 and other proteins), but not a lower stringency assay that would be expected to also detect functional, actin-bound TRIOBP-1. Previously, we have also determined that a region of 25 amino acids in the center of this protein is critical for its ability to form aggregates. Here we attempt to refine this further, through the expression of various truncated mutant TRIOBP-1 vectors in neuroblastoma cells and examining their aggregation. In this way, it was possible to narrow down the aggregation-critical region of TRIOBP-1 to just 8 amino acids (333-340 of the 652 amino acid-long TRIOBP-1). Surprisingly our results suggested that a second section of TRIOBP-1 is also capable of independently inducing aggregation: the optionally expressed 59 amino acids at the extreme N-terminus of the protein. As a result, the 597 amino acid long version of TRIOBP-1 (also referred to as "Tara" or "TAP68") has reduced potential to form aggregates. The presence of insoluble TRIOBP-1 in brain samples from patients, combined with insight into the mechanism of aggregation of TRIOBP-1 and generation of an aggregation-resistant mutant TRIOBP-1 that lacks both these regions, will be of significant use in further investigating the mechanism and consequences of TRIOBP-1 aggregation in major mental illness.

Protein Aggregation of NPAS3, Implicated in Mental Illness, Is Not Limited to the V304I Mutation.

An emerging phenomenon in our understanding of the pathophysiology of mental illness is the idea that specific proteins may form insoluble aggregates in the brains of patients, in partial analogy to similar proteinopathies in neurodegenerative diseases. Several proteins have now been detected as forming such aggregates in the brains of patients, including DISC1, dysbindin-1 and TRIOBP-1. Recently, neuronal PAS domain protein 3 (NPAS3), a known genetic risk factor for schizophrenia, was implicated through a V304I point mutation in a family with major mental illness. Investigation of the mutation revealed that it may lead to aggregation of NPAS3. Here we investigated NPAS3 aggregation in insular cortex samples from 40 individuals, by purifying the insoluble fraction of these samples and testing them by Western blotting. Strikingly, full-length NPAS3 was found in the insoluble fraction of 70% of these samples, implying that aggregation is far more widely spread than can be accounted for by this rare mutation. We investigated the possible mechanism of aggregation further in neuroblastoma cells, finding that oxidative stress plays a larger role than the V304I mutation. Finally, we tested to see if NPAS3 aggregation could also be seen in blood serum, as a more accessible tissue than the human brain for future diagnosis. While no indication of NPAS3 aggregation was seen in the serum, soluble NPAS3 was detected, and was more prevalent in patients with schizophrenia than in those with major depressive disorder or controls. Aggregation of NPAS3 therefore appears to be a widespread and multifactorial phenomenon. Further research is now needed to determine whether it is specifically enhanced in schizophrenia or other mental illnesses.

The TRIOBP Isoforms and Their Distinct Roles in Actin Stabilization, Deafness, Mental Illness, and Cancer.

The TRIOBP (TRIO and F-actin Binding Protein) gene encodes multiple proteins, which together play crucial roles in modulating the assembly of the actin cytoskeleton. Splicing of the TRIOBP gene is complex, with the two most studied TRIOBP protein isoforms sharing no overlapping amino acid sequence with each other. TRIOBP-1 (also known as TARA or TAP68) is a mainly structured protein that is ubiquitously expressed and binds to F-actin, preventing its depolymerization. It has been shown to be important for many processes including in the cell cycle, adhesion junctions, and neuronal differentiation. TRIOBP-1 has been implicated in schizophrenia through the formation of protein aggregates in the brain. In contrast, TRIOBP-4 is an entirely disordered protein with a highly specialized expression pattern. It is known to be crucial for the bundling of actin in the stereocilia of the inner ear, with mutations in it causing severe or profound hearing loss. Both of these isoforms are implicated in cancer. Additional longer isoforms of TRIOBP exist, which overlap with both TRIOBP-1 and 4. These appear to participate in the functions of both shorter isoforms, while also possessing unique functions in the inner ear. In this review, the structures and functions of all of these isoforms are discussed, with a view to understanding how they operate, both alone and in combination, to modulate actin and their consequences for human illness.