Loss of Hyaluronan and Proteoglycan Link Protein-1 Induces Tumorigenesis in Colorectal Cancer

Colorectal cancer (CRC) is the third most common diagnosed cancer worldwide, but there are no effective cures for it. Hyaluronan and proteoglycan link protein-1 (HAPLN1) is a component of the extracellular matrix (ECM) proteins and involved in the tumor environment in the colon. Transforming growth factor (TGF)-β is a key cytokine that regulates the deposition of ECM proteins in CRC. However, the role of HAPLN1 in TGF-β contributions to CRC remains unknown. We found that the mRNA expression of HAPLN1 was decreased in tumors from CRC patients compared with healthy controls and normal tissue adjacent to the tumor using two existing microarray datasets. This was validated at the protein level by tissue array from CRC patients (n = 59). HAPLN1 protein levels were also reduced in human CRC epithelial cells after 24 h of TGF-β stimulation, and its protein expression correlated with type I collagen alpha-1 (COL1A1) in CRC. Transfection of HAPLN1 overexpression plasmids into these cells increased protein levels but reduced COL1A1 protein, tumor growth, and cancer cell migration. TGF-β stimulation increased Smad2/3, p-Smad2/3, Smad4, and E-adhesion proteins; however, HAPLN1 overexpression restored these proteins to baseline levels in CRC epithelial cells after TGF-β stimulation. These findings suggest that HAPLN1 regulates the TGF-β signaling pathway to control collagen deposition via the TGF-β signaling pathway and mediates E-adhesion to control tumor growth. Thus, treatments that increase HAPLN1 levels may be a novel therapeutic option for CRC.

Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) Promotes Viability of Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Background To investigate HAPLN1 contribution to the viability of RA-FLSs and identify its potential role in RA pathogenesis. Methods Plasma levels and synovial expression of HAPLN1 were compared between healthy controls, and osteoarthritis (OA) and RA patients. Proliferation and migration of RA-FLSs transfected with siHAPLN1, HAPLN1OE (over-expression vector) and respective controls or treated with rHAPLN1 were measured by MTT and CCK8 assays as well as wound healing and transwell assays. RT-qPCR and automated WB analysis were used to compare the expression of AMPK-ɑ, TNF-ɑ, TGF-β, ACAN, MMPs, Cyclin-D1 and Ki-67 after siHAPLN1 or HAPLN1OE transfection. Proteomics and mRNA-seq analysis was done to study the differentially expressed proteins/genes after siHAPLN1 or rHAPLN1 treatment. Results Expression of HAPLN1 was increased in the plasma samples and synovium tissues of RA patients. HAPLN1OE transfected or rHAPLN1 treated RA-FLSs showed an increased proliferation capacity. However, Si-HAPLN1 has failed to affect the viability of RA-FLSs, though it decreased the migration ability of these cells. On the other hand, HAPLN1OE or rHAPLN1 had inhibitory effect on migration. Both si-HAPLN1 and HAPLN1OE treated RA-FLSs had down-regulated AMPK-ɑ gene expression, while protein level was found to be up-regulated. Furthermore, si-HAPLN1 has down-regulated TNF-ɑ, MMPs, IL-6, TGF-β, fibronectin and ACAN levels, while HAPLN1OE has up-regulated the levels of TNF-ɑ, MMPs, IL-6 and ACAN. Proteomics and mRNA-Seq analysis demonstrated HAPLN1 function in the activation of inflammation, proliferation, increased cell adhesion and strengthening of ECM function. Conclusons: HAPLN1 accelerated the proliferation of RA-FLSs but inhibited its migration ability. HAPLN1 network was found to be mainly involved in the activation of inflammation, cell proliferation and increased cell adhesion.

Assessment of Possible Contributions of Hyaluronan and Proteoglycan Binding Link Protein 4 to Differential Perineuronal Net Formation at the Calyx of Held

The calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of Hapln4 exhibited a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the neuropil surrounding the whole calyx of Held terminals. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil, together with HAPLN1 and tenascin-R, in both gene knockout (KO) and wild-type (WT) mice. An in situ proximity ligation assay demonstrated the molecular association of brevican with HAPLN4 in WT and HAPLN1 in gene KO mice. Further elucidation of the roles of HAPLN4 may highlight the developmental and physiological importance of PNN formation in the calyx of Held.

The Role of Protein Persulfidation in Brain Aging and Neurodegeneration

Hydrogen sulfide (H2S), originally considered a toxic gas, is now a recognized gasotransmitter. Numerous studies have revealed the role of H2S as a redox signaling molecule that controls important physiological/pathophysiological functions. The underlying mechanism postulated to serve as an explanation of these effects is protein persulfidation (P-SSH, also known as S-sulfhydration), an oxidative posttranslational modification of cysteine thiols. Protein persulfidation has remained understudied due to its instability and chemical reactivity similar to other cysteine modifications, making it very difficult to selectively label. Recent developments of persulfide labeling techniques have started unraveling the role of this modification in (patho)physiology. PSSH levels are important for the cellular defense against oxidative injury, albeit they decrease with aging, leaving proteins vulnerable to oxidative damage. Aging is one of the main risk factors for many neurodegenerative diseases. Persulfidation has been shown to be dysregulated in Parkinson's, Alzheimer's, Huntington's disease, and Spinocerebellar ataxia 3. This article reviews the latest discoveries that link protein persulfidation, aging and neurodegeneration, and provides future directions for this research field that could result in development of targeted drug design.

Weakening of Interaction Networks with Aging in Tip-Link Protein Induces Hearing Loss

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing-loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among β-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in β-strand-rich proteins.

A Role for HAPLN1 During Phenotypic Modulation of Human Lung Fibroblasts In Vitro

Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan, which facilitate proliferation of fibroblasts and their conversion to myofibroblasts. However, the role of HAPLN1 in these events has not been studied. Using immunocytochemistry, cellular and ECM locations of HAPLN1 were evaluated in cultured human lung fibroblasts during proliferation and conversion to myofibroblasts. HAPLN1 localized to pericellular matrices, associating with both versican and hyaluronan in the ECM and on the cell surface. Nuclear and total HAPLN1 immunostaining increased after myofibroblast induction. Confocal microscopy showed HAPLN1 predominant in the ECM under cells while versican predominated above cells. Versican and HAPLN1 were also juxtaposed in columnar inclusions in the cytoplasm and nucleus. Nuclear HAPLN1 staining in interphase cells redistributed to the cytosol during mitosis. In the absence of TGF-β1, addition of exogenous bovine HAPLN1 (together with aggrecan G1) facilitated myofibroblast formation, as seen by significant upregulation of α-smooth muscle actin (SMA) staining, while adding full-length bovine versican had no effect. Increased compaction of hyaluronan-rich ECM suggests that HAPLN1 plus G1 addition affects hyaluronan networks and myofibroblast formation. These observations demonstrate changes in both extracellular and intracellular localization of HAPLN1 during fibroblast proliferation and myofibroblast conversion suggesting a possible role in fibrotic remodeling:

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes of Saccharomyces cerevisiae Bloom’s Helicase Homolog Sgs1 SUMOylation-Associated Mutants

The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21-mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

Quantitative Proteomics of All 14 Renal Tubule Segments in Rat

BackgroundPrevious research has used RNA sequencing in microdissected kidney tubules or single cells isolated from the kidney to profile gene expression in each type of kidney tubule epithelial cell. However, because proteins, not mRNA molecules, mediate most cellular functions, it is desirable to know the identity and amounts of each protein species to understand function. Recent improvements in the sensitivity of mass spectrometers offered us the ability to quantify the proteins expressed in each of 14 different renal tubule segments from rat.MethodsWe manually dissected kidney tubules from rat kidneys and subjected samples to protein mass spectrometry. We used the “proteomic ruler” technique to estimate the number of molecules of each protein per cell.ResultsOver the 44 samples analyzed, the average number of quantified proteins per segment was 4234, accounting for at least 99% of protein molecules in each cell. We have made the data publicly available online at the Kidney Tubule Expression Atlas website (https://esbl.nhlbi.nih.gov/KTEA/). Protein abundance along the renal tubule for many commonly studied water and solute transport proteins and metabolic enzymes matched expectations from prior localization studies, demonstrating the overall reliability of the data. The site features a “correlated protein” function, which we used to identify cell type–specific transcription factors expressed along the renal tubule.ConclusionsWe identified and quantified proteins expressed in each of the 14 segments of rat kidney tubules and used the proteomic data that we obtained to create an online information resource, the Kidney Tubule Expression Atlas. This resource will allow users throughout the world to browse segment-specific protein expression data and download them for their own research.

Distribution and classification of the extracellular matrix in the olfactory bulb

Extracellular matrix (ECM) became an important player over the last few decades when studying the plasticity and regeneration of the central nervous system. In spite of the established role of ECM in these processes throughout the central nervous system (CNS), only few papers were published on the ECM of the olfactory system, which shows a lifelong plasticity, synaptic remodeling and postnatal neurogenesis. In the present study, we have described the localization and organization of major ECM molecules, the hyaluronan, the lecticans, tenascin-R and HAPLN1 link protein in the olfactory bulb (OB) of the rat. We detected all of these molecules in the OB showing differences in the molecular composition, staining intensity, and organization of ECM between the layers and in some cases within a single layer. One of the striking features of ECM staining pattern in the OB was that the reactions are shown dominantly in the neuropil, the PNNs were found rarely and they exhibited thin or diffuse appearance Similar organization was shown in human and mice samples. As the PNN limits the neural plasticity, its rare appearance may be related to the high degree of plasticity in the OB.

Asymmetric Hapln1a drives regionalised cardiac ECM expansion and promotes heart morphogenesis during zebrafish development

The mature vertebrate heart develops from a simple linear cardiac tube during early development through a series of highly asymmetric morphogenetic processes including cardiac looping and chamber ballooning. While the directionality of heart morphogenesis is partly controlled by embryonic laterality signals, previous studies have suggested that these extrinsic laterality cues interact with tissue-intrinsic signals in the heart to ensure robust asymmetric cardiac morphogenesis. Using live in vivo imaging of zebrafish embryos we describe a left-sided, chamber-specific expansion of the extracellular matrix (ECM) between the myocardium and endocardium at early stages of heart morphogenesis. We use Tomo-seq, a spatial transcriptomic approach, to identify transient and regionalised expression of hyaluronan and proteoglycan link protein 1a (hapln1a), encoding an ECM cross-linking protein, in the heart tube prior to cardiac looping overlapping with regionalised ECM expansion. Loss- and gain-of-function experiments demonstrate that regionalised Hapln1a promotes heart morphogenesis through regional modulation of ECM thickness in the heart tube. Finally, we show that while induction of asymmetric hapln1a expression is independent of embryonic left-right asymmetry, these laterality cues are required to orient the hapln1a-expressing cells asymmetrically along the left-right axis of the heart tube.Together, we propose a model whereby laterality cues position hapln1a expression on the left of the heart tube, and this asymmetric Hapln1a deposition drives ECM asymmetry and subsequently promotes robust asymmetric cardiac morphogenesis.