Viraj Ichhaporia

Abstract:

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation… Show more

Abstract:

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1′s function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1′s functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1′s NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS. Show less

SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A–D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss… Show more

SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A–D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss of SEC24C. Conditional KO of Sec24c in neural progenitors during embryogenesis caused perinatal mortality and microcephaly, with activation of the unfolded protein response and apoptotic cell death of postmitotic neurons in the murine cerebral cortex. The cell-autonomous function of SEC24C in postmitotic neurons was further highlighted by the loss of cell viability caused by disrupting Sec24c expression in forebrain neurons of mice postnatally and in differentiated neurons derived from human induced pluripotent stem cells. The neuronal cell death associated with Sec24c deficiency was rescued in knockin mice expressing Sec24d in place of Sec24c. These data suggest that SEC24C is a major cargo adapter for COPII-dependent transport in postmitotic neurons in developing and adult brains and that its functions overlap at least partially with those of SEC24D in mammals. Show less

ABSTRACT:
Mutations in SIL1, a co-factor for the endoplasmic reticular (ER) chaperone BiP, cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive disorder. Using a mouse model, we characterized molecular aspects of the progressive myopathy associated with MSS. Proteomic-profiling of quadriceps at the onset of myopathy revealed that SIL1 deficiency affected multiple pathways critical to muscle physiology. We observed an increase in ER chaperones prior to the onset of muscle weakness… Show more

ABSTRACT:
Mutations in SIL1, a co-factor for the endoplasmic reticular (ER) chaperone BiP, cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive disorder. Using a mouse model, we characterized molecular aspects of the progressive myopathy associated with MSS. Proteomic-profiling of quadriceps at the onset of myopathy revealed that SIL1 deficiency affected multiple pathways critical to muscle physiology. We observed an increase in ER chaperones prior to the onset of muscle weakness, which was complemented by up-regulation of multiple components of cellular protein degradation pathways. These responses were inadequate to maintain normal expression of secretory pathway proteins, including Insulin and IGF-1 receptors. There was a paradoxical enhancement of downstream PI3K-AKT signaling and glucose uptake in SIL1-disrupted skeletal muscles, all of which were insufficient to maintain skeletal muscle mass. Together, these data reveal a disruption in ER homeostasis upon SIL1 loss, which is countered by multiple compensatory responses that are ultimately unsuccessful, leading to trans-organellar proteostasis collapse and myopathy.

KEYWORDS:
Endoplasmic reticulum; Marinesco-Sjögren syndrome; Myopathy; PI3K-AKT-mTOR signaling; Proteostasis collapse; SIL1 Show less

Abstract:
Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and… Show more

Abstract:
Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two-dimensional liquid chromatography, TiO2-mediated phosphopeptide enrichment, high-resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens. Show less

Abstract: Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco-Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multi-system defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1Gt mouse phenocopies several pathologies associated with MSS, although their ability to assemble and secrete antibodies, the best characterized… Show more

Abstract: Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco-Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multi-system defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1Gt mouse phenocopies several pathologies associated with MSS, although their ability to assemble and secrete antibodies, the best characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1Gt mouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS stimulated splenic B cells from Sil1Gt mice. ER chaperones were expressed at the same level in Sil1WT and Sil1Gt mice indicating that there was no evident compensation for the disruption of Sil1. Lastly, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production. Show less