Biology subjects

Bennett, D. C.

Publications and source records attributed to Bennett, D. C..

3 recordsLinked to original sources

bioRxiv2021

Enhancing top-down proteomics of brain tissue with FAIMS

Proteomic investigations of Alzheimers and Parkinsons disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a proteins "intact" state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Utilizing a high complexity sample derived from Alzheimers disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at -50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 {+/-} 17, n = 3), compared to without (754 {+/-} 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (A{beta}) proteoforms, including the aggregation-prone A{beta}1-42 variant which is strongly linked to Alzheimers disease.

biochemistry↗

bioRxiv2020

Loss of Kallmann syndrome-associated gene WDR11 disrupts primordial germ cell development by affecting canonical and non-canonical Hedgehog signalling

Mutations of WDR11 are associated with Kallmann syndrome (KS) and congenital hypogonadotrophic hypogonadism (CHH), typically caused by defective functions of gonadotrophin-releasing hormone (GnRH) neurones in the brain. We previously reported that Wdr11 knockout mice show profound infertility with significantly fewer germ cells present in the gonads. To understand the underlying mechanisms mediated by WDR11 in these processes, we investigated the effects of Wdr11 deletion on primordial germ cell (PGC) development. Using live-tracking of PGCs and primary co-cultures of genital ridges (GR), we demonstrated that Wdr11-deficient embryos contained reduced numbers of PGCs which had delayed migration due to significantly decreased proliferation and motility. We found primary cilia-dependent canonical Hedgehog (Hh) signalling was required for proliferation of the somatic mesenchymal cells of GR, while primary cilia-independent non-canonical Hh signalling mediated by Ptch2/Gas1 and downstream effectors Src and Creb was required for PGC proliferation and migration, which was disrupted by the loss of function mutations of WDR11. Therefore, canonical and non-canonical Hh signalling are differentially involved in the development of somatic and germ cell components of the gonads, and WDR11 is required for both of these pathways operating in parallel in GR and PGCs, respectively, during normal PGC development. Our study provides a mechanistic link between the development of GnRH neurones and germ cells mediated by WDR11, which may underlie some cases of KS/CHH and ciliopathies.

genetics↗

bioRxiv2020

SLC45A2 protein stability and regulation of melanosome pH determine melanocyte pigmentation

SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations and polymorphisms cause oculocutaneous albinism (OCA) and pigmentation variation, but neither SLC45A2 localization and function nor how gene variants affect these properties are known. We show that SLC45A2 localizes to mature melanosomes that only partially overlap with a cohort expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that, like OCA2, SLC45A2 in melanocytes de-acidifies maturing melanosomes to support melanin synthesis. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 functions later during melanosome maturation than OCA2, and that OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. The light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes because of low level expression in melanosomes due to rapid proteasome-independent degradation. Our data indicate that SLC45A2 maintains melanosome neutralization - initially orchestrated by transient OCA2 activity - to support melanization at late stages of melanosome maturation, and that a common variant imparts reduced activity due to protein instability.

cell biology↗