Biomedical subjects

Michal L Ram

Publications and source records attributed to Michal L Ram.

4 recordsLinked to original sources

PubMed2006

Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ.

Cardiac calsequestrin (CSQ) is a protein that traffics to and concentrates inside sarcoplasmic reticulum (SR) terminal cisternae, a protein secretory compartment of uncertain origin. To investigate trafficking of CSQ within standard ER compartments, we expressed CSQ in nonmuscle cell lines and examined its localization by immunofluorescence and its molecular structure from the mass spectrum of total cellular CSQ. In all cells examined, CSQ was a highly phosphorylated protein with a glycan structure predictive of ER-retained proteins: Man9,8GlcNAc2 lacking terminal GlcNAc. Immunostaining was restricted to polymeric ER cisternae. Secretory pathway disruption by brefeldin A and thapsigargin led to altered CSQ glycosylation and phosphorylation consistent with post-ER trafficking. When epitope-tagged forms of CSQ were expressed in the same cells, mannose trimming of CSQ glycans was far more extensive, and C-terminal phosphorylation sites were nearly devoid of phosphate, in complete contrast to the highly phosphorylated wild-type protein that concentrates in all cells tested. Epitope-tagged CSQ also showed a reduced ER staining compared to wild-type protein, with significant staining in juxta-Golgi compartments. Loss of ER retention due to epitope tags or thapsigargin and resultant changes in protein structure or levels of bound Ca(2+) point to CSQ polymerization as an ER/SR retention mechanism.

Animals↗

PubMed2004

Calsequestrin mutant D307H exhibits depressed binding to its protein targets and a depressed response to calcium.

OBJECTIVE: A point mutation in human cardiac calsequestrin (CSQ-D307H) is responsible for a form of polymorphic ventricular tachycardia (PVT). When overexpressed in heart cells, the mutated CSQ leads to diminished Ca(2+) transients, consistent with defective regulation of intralumenal sarcoplasmic reticulum (SR) Ca(2+). METHODS: To analyze the D307H mutant and determine whether the D307H mutation results in loss of normal protein-protein interactions, we prepared recombinant human wild-type (WT) and D307H forms of CSQ in mammalian cells. RESULTS: Although we found the two proteins to undergo similar glycosylation and phosphorylation, we discovered that Ca(2+)-dependent binding of the D307H mutant to both triadin-1 and junctin was reduced by greater than 50% compared to WT. Reduced binding of the D307H mutant CSQ to target proteins was similar throughout a complete range of Ca(2+) concentrations. To investigate the mechanism of reduced Ca(2+)-dependent binding, Ca(2+)-dependent changes in intrinsic fluorescence emission for the two protein forms were compared. Intrinsic fluorescence of the D307H mutant was highly reduced, reflecting significant alteration in the tertiary protein structure. Moreover, the changes in fluorescence caused by increasing the Ca(2+) concentration were very significantly blunted, indicating that the Ca(2+)-dependent conformational change was virtually lost. CONCLUSIONS: We conclude that the point mutation D307H leads to a profoundly altered conformation that no longer responds normally to Ca(2+) and fails to bind normally to triadin and junctin.

Animals↗

PubMed2004

Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart.

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.

Adenoviridae↗

PubMed2002

Mass spectrometry of cardiac calsequestrin characterizes microheterogeneity unique to heart and indicative of complex intracellular transit.

Cardiac calsequestrin concentrates in junctional sarcoplasmic reticulum in heart and skeletal muscle cells by an undefined mechanism. During transit through the secretory pathway, it undergoes an as yet uncharacterized glycosylation and acquires phosphate on CK2-sensitive sites. In this study, we have shown that active calsequestrin phosphorylation occurred in nonmuscle cells as well as muscle cells, reflecting a widespread cellular process. To characterize this post-translational modification and resolve individual molecular mass species, we subjected purified calsequestrin to mass spectrometry using electrospray ionization. Mass spectra showed that calsequestrin glycan structure in nonmuscle cells was that expected for an endoplasmic reticulum-localized glycoprotein and showed that each glycoform existed as four mass peaks representing molecules that also had 0-3 phosphorylation sites occupied. In heart, mass peaks indicated carbohydrate modifications characteristic of transit through Golgi compartments. Phosphorylation did not occur on every glycoform present, suggesting a far more complex movement of calsequestrin molecules in heart cells. Significant amounts of calsequestrin contained glycan with only a single mannose residue, indicative of a novel post-endoplasmic reticulum mannosidase activity. In conclusion, glyco- and phosphoforms of calsequestrin chart a complex cellular transport in heart, with calsequestrin following trafficking pathways not present or not accessible to the same molecules in nonmuscle.

Animals↗