scholarly journals ­­BVES Is a Novel Interactor of ANO5 and Regulates Myoblast Differentiation

2021 ◽  
Author(s):  
Haiwen Li ◽  
Li Xu ◽  
Yandi Gao ◽  
Yuanbojiao Zuo ◽  
Zuocheng Yang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Interacting Proteins ◽  
Cellular Functions ◽  
C2c12 Myoblasts ◽  
Interacting Protein ◽  
C Terminus ◽  
N Terminus ◽  
Labeling Approach

Abstract Background: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins specifically from ANO5-BioID2 samples but not BioID2 fused with ANO6 or MG53, including BVES and POPDC3. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with BVES through its C-terminus. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions:Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

scholarly journals BVES is a novel interactor of ANO5 and regulates myoblast differentiation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Haiwen Li ◽  
Li Xu ◽  
Yandi Gao ◽  
Yuanbojiao Zuo ◽  
Zuocheng Yang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Interacting Proteins ◽  
Cellular Functions ◽  
C2c12 Myoblasts ◽  
Interacting Protein ◽  
C Terminus ◽  
N Terminus ◽  
Labeling Approach

Abstract Background Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

scholarly journals An mDia1-INF2 formin activation cascade facilitated by IQGAP1 regulates stable microtubules in migrating cells

2016 ◽  
Vol 27 (11) ◽  
pp. 1797-1808 ◽  
Author(s):  
Francesca Bartolini ◽  
Laura Andres-Delgado ◽  
Xiaoyi Qu ◽  
Sara Nik ◽  
Nagendran Ramalingam ◽  
...  
Keyword(s):  
Lysophosphatidic Acid ◽  
Dependent Manner ◽  
Common Pathway ◽  
Interacting Protein ◽  
C Terminus ◽  
N Terminus ◽  
In Series ◽  
Activation Cascade ◽  
Unique Mechanism ◽  
Migrating Cells

Multiple formins regulate microtubule (MT) arrays, but whether they function individually or in a common pathway is unknown. Lysophosphatidic acid (LPA) stimulates the formation of stabilized detyrosinated MTs (Glu MTs) in NIH3T3 fibroblasts through RhoA and the formin mDia1. Here we show that another formin, INF2, is necessary for mDia1-mediated induction of Glu MTs and regulation of MT dynamics and that mDia1 can be bypassed by activating INF2. INF2 localized to MTs after LPA treatment in an mDia1-dependent manner, suggesting that mDia1 regulates INF2. Mutants of either formin that disrupt their interaction failed to rescue MT stability in cells depleted of the respective formin, and the mDia1-interacting protein IQGAP1 regulated INF2’s localization to MTs and the induction of Glu MTs by either formin. The N-terminus of IQGAP1 associated with the C-terminus of INF2 directly, suggesting the possibility of a tripartite complex stimulated by LPA. Supporting this, the interaction of mDia1 and INF2 was induced by LPA and dependent on IQGAP1. Our data highlight a unique mechanism of formin action in which mDia1 and INF2 function in series to stabilize MTs and point to IQGAP1 as a scaffold that facilitates the activation of one formin by another.

scholarly journals Escherichia coli l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry

1997 ◽  
Vol 323 (3) ◽  
pp. 661-669 ◽  
Author(s):  
Manoj K. RAMJEE ◽  
Ulrich GENSCHEL ◽  
Chris ABELL ◽  
Alison G. SMITH
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Electrospray Mass Spectrometry ◽  
Elevated Temperatures ◽  
Gene Encoding ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Terminal Amino ◽  
Α Subunits

The Escherichia coli panD gene, encoding l-aspartate-α-decarboxylase, was cloned by PCR, and shown to complement apanD mutant defective in β-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the π-protein), which is proteolytically cleaved at a specific X–Ser bond to produce a β-subunit with XOH at its C-terminus and an α-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed π-subunit (of 13.8 kDa), with a small proportion of the α- and β-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the α-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151±16 μM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

scholarly journals Osteocalcin Induces Proliferation via Positive Activation of the PI3K/Akt, P38 MAPK Pathways and Promotes Differentiation Through Activation of the GPRC6A-ERK1/2 Pathway in C2C12 Myoblast Cells

2017 ◽  
Vol 43 (3) ◽  
pp. 1100-1112 ◽  
Author(s):  
Suifeng Liu ◽  
Feng Gao ◽  
Lei Wen ◽  
Min Ouyang ◽  
Yi Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
P38 Mapk ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
C2c12 Myoblasts ◽  
Mapk Phosphorylation ◽  
Mapk Pathways ◽  
Myoblast Cells

Background/Aims: Sarcopenia is characterized by an age-related decline in skeletal muscle plus low muscle strength and/or physical performance. Despite the clinical significance of sarcopenia, the molecular pathways underlying sarcopenia remain elusive. The recent demonstration that undercarboxylated osteocalcin (ucOC) favours muscle function related to insulin sensitivity and glucose metabolism raises the question of whether this hormone may also regulate muscle mass. The present study explored the promotive effects of ucOC in proliferation and differentiation processes of C2C12 myoblasts as well as the possible signalling pathways involved. Methods: The effects of exogenous ucOC on C2C12 myoblasts proliferation were assessed using CCK8 and immunohistological staining assays. C2C12 cells were pretreated with PI3K/Akt or P38 MAPK inhibitors to investigate the possible involvement of the PI3K/Akt and P38 MAPK pathways in proliferation. The levels of Akt, phosphorylated-Akt (p-Akt), P38, and phosphorylated-P38 (p-P38) were measured by Western Blotting. The effects of ucOC on myoblast differentiation were quantified by morphological analysis. A silencing experiment was conducted in which the expression of GPRC6A in C2C12 myoblasts was modified. The expression of GPRC6A, myosin heavy chain (MyHC) and the related ERK1/2 signalling pathway in C2C12 myoblasts were monitored by qRT-PCR and Western Blotting. Results: We showed that treatment with exogenous ucOC stimulated the priming of C2C12 myoblasts proliferation. Inhibition of Akt phosphorylation by wortmannin or inhibition of P38 MAPK phosphorylation by SB203580 decreased C2C12 cell proliferation. Wortmannin also reduced P38 MAPK phosphorylation, whereas SB203580 did not affect Akt activation. Furthermore, ucOC promoted C2C12 myoblast differentiation. Inhibition of ERK1/2 phosphorylation with U0126 decreased C2C12 cell differentiation. Finally, GPRC6A expression was substantially increased after ucOC treatment of C2C12 cells. GPRC6A silencing inhibited Akt, P38 MAPK phosphorylation in C2C12 cells, and ERK1/2 phosphorylation in C2C12 myotubes; GPRC6A silencing also decreased cell proliferation, decreased cell differentiation, and downregulated MyHC expression. Conclusions: The present data suggest that ucOC induces myoblast proliferation via sequential activation of the PI3K/Akt and p38 MAPK pathways in C2C12 myoblast cells. Moreover, ucOC enhances myogenic differentiation via a mechanism involving GPRC6A-ERK1/2 signalling.

scholarly journals Ganoderma microsporum immunomodulatory protein, GMI, promotes C2C12 myoblast differentiation in vitro via upregulation of Tid1 and STAT3 acetylation

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244791
Author(s):  
Wan-Huai Teo ◽  
Jeng-Fan Lo ◽  
Yu-Ning Fan ◽  
Chih-Yang Huang ◽  
Tung-Fu Huang
Keyword(s):  
Myogenic Differentiation ◽  
Atp Synthesis ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Muscle Loss ◽  
C2c12 Myoblasts ◽  
Immunomodulatory Protein ◽  
Pre Treatment

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it’s crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3.

scholarly journals Internal Fragments Generated by Electron Ionization Dissociation Enhances Protein Top-down Mass Spectrometry

2020 ◽  
Author(s):  
Muhammad Zenaidee ◽  
Carter Lantz ◽  
Taylor Perkins ◽  
Janine Fu ◽  
Wonhyuek Jung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Electron Ionization ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Top Down ◽  
C Terminus ◽  
N Terminus ◽  
Product Ions ◽  
Protein Fragmentation

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that do not contain the C-terminus or N-terminus of the protein. Conventionally, internal fragments have been disregarded as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50% to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments.

scholarly journals Dimerization of ubiquilin is dependent upon the central region of the protein: evidence that the monomer, but not the dimer, is involved in binding presenilins

2006 ◽  
Vol 399 (3) ◽  
pp. 397-404 ◽  
Author(s):  
Diana L. Ford ◽  
Mervyn J. Monteiro
Keyword(s):  
Central Region ◽  
Regulatory Mechanism ◽  
Active Form ◽  
Cellular Proteins ◽  
Interacting Protein ◽  
C Terminus ◽  
N Terminus ◽  
The Stability ◽  
Insight Into

Ubiquilin proteins have been shown to interact with a wide variety of other cellular proteins, often regulating the stability and degradation of the interacting protein. Ubiquilin contains a UBL (ubiquitin-like) domain at the N-terminus and a UBA (ubiquitin-associated) domain at the C-terminus, separated by a central region containing Sti1-like repeats. Little is known about regulation of the interaction of ubiquilin with other proteins. In the present study, we show that ubiquilin is capable of forming dimers, and that dimerization requires the central region of ubiquilin, but not its UBL or the UBA domains. Furthermore, we provide evidence suggesting that monomeric ubiquilin is likely to be the active form that is involved in binding presenilin proteins. Our results provide new insight into the regulatory mechanism underlying the interaction of ubiquilin with presenilins.

scholarly journals Muscle Enriched Lamin Interacting Protein (Mlip) Binds Chromatin and Is Required for Myoblast Differentiation

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 615
Author(s):  
Elmira Ahmady ◽  
Alexandre Blais ◽  
Patrick G. Burgon
Keyword(s):  
Protein Complexes ◽  
Myogenic Differentiation ◽  
Direct Interaction ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Mobility Shift ◽  
Interacting Protein ◽  
Gel Mobility Shift ◽  
Genomic Regions ◽  
Myogenic Program

Muscle-enriched A-type lamin-interacting protein (Mlip) is a recently discovered Amniota gene that encodes proteins of unknown biological function. Here we report Mlip’s direct interaction with chromatin, and it may function as a transcriptional co-factor. Chromatin immunoprecipitations with microarray analysis demonstrated a propensity for Mlip to associate with genomic regions in close proximity to genes that control tissue-specific differentiation. Gel mobility shift assays confirmed that Mlip protein complexes with genomic DNA. Blocking Mlip expression in C2C12 myoblasts down-regulates myogenic regulatory factors (MyoD and MyoG) and subsequently significantly inhibits myogenic differentiation and the formation of myotubes. Collectively our data demonstrate that Mlip is required for C2C12 myoblast differentiation into myotubes. Mlip may exert this role as a transcriptional regulator of a myogenic program that is unique to amniotes.

scholarly journals Internal Fragments Generated by Electron Ionization Dissociation Enhances Protein Top-down Mass Spectrometry

2020 ◽  
Author(s):  
Muhammad Zenaidee ◽  
Carter Lantz ◽  
Taylor Perkins ◽  
Janine Fu ◽  
Wonhyuek Jung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Electron Ionization ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Top Down ◽  
C Terminus ◽  
N Terminus ◽  
Product Ions ◽  
Protein Fragmentation

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that do not contain the C-terminus or N-terminus of the protein. Conventionally, internal fragments have been disregarded as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50% to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments.

scholarly journals Internal Fragments Generated by Electron Ionization Dissociation Enhances Protein Top-down Mass Spectrometry

2020 ◽  
Author(s):  
Muhammad Zenaidee ◽  
Carter Lantz ◽  
Taylor Perkins ◽  
Janine Fu ◽  
Wonhyuek Jung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer Dissociation ◽  
Electron Ionization ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Top Down ◽  
C Terminus ◽  
N Terminus ◽  
Product Ions ◽  
Protein Fragmentation

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation (ECD) and electron transfer dissociation (ETD) are widely used for these types of analysis, however these fragmentation methods can be inefficient due to the low energy electrons fragmenting the protein without the dissociation products; that is no detection of fragments formed. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (> 20 eV) has been shown to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that do not contain the C-terminus or N-terminus of the protein. Conventionally, internal fragments have been disregarded as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50% to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. By including internal fragments in the data analysis, previously unassigned peaks can be readily and accurately assigned to enhance the efficiencies of top-down protein sequencing experiments.

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