Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection of Colorectal Cancer

Colorectal cancer (CRC) is a common and treatable disease if diagnosed early. Current population screening programs are suboptimal, and consequently, there is a need for the development of new methodologies for early diagnosis of CRC. In the past 10 years, unprecedented technological advancements in the field of mass spectrometry (MS)-based proteomics have progressively increased the sophistication and utility of these investigations, leading to the draft mapping of the human proteome. These exciting studies have shaped our mechanistic understanding of the human genome and begun to provide us with a suite of novel biomarkers to predict the onset, progression and severity of many debilitating diseases. Thus, sophisticated MS workflows coupled with revolutionary protein quantification techniques hold promise for the field of MS-based plasma proteomics, particularly valuable in the context of early stage identification of curable CRC. However, within the last 40 years, no new plasma protein biomarkers of CRC have been translated into clinical practice. Here. we discuss the application of proteomic technologies within the field of CRC, highlighting contemporary MS-based plasma proteomic strategies that could be exploited to deliver on the promise of a panel of sensitive and specific plasma-based biomarkers with which to non-invasively detect early stage CRC

Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions

Abstract Background The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. Results Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. Conclusion Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation

Preclinical and clinical evaluation of German-sourced ONC201 for the treatment of H3K27M-mutant diffuse intrinsic pontine glioma

Background Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound-ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. Methods Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017-2020) was analyzed. Results GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). Conclusions This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy

The molecular basis of sperm - oocyte interactions

Research Doctorate - Doctor of Philosophy (PhD)The remarkable cellular communication events that characterise the highly species specific interactions observed during the ontogeny of mammalian fertilization, represent some of the most intriguing in all of biology. Given the 60 years or so of research conducted to elucidate the precise mechanisms that underpin these interactions, it is surprising that they still remain largely unknown. This can be mostly attributed to the unique luminal environment in which the sperm reside following insemination and the direct effects that these fluids have on their functionality. Although immense controversy surrounds the precise ligand responsible for the spermatozoas binding to the oocyte’s zona pellucida, considerable contention is also afforded to the mechanism by which they bind. A number of landmark papers have recently emerged to suggest that these initial binding events may be facilitated by the formation and presentation of multimeric zona pellucida receptor complexes on the sperm surface during their terminal maturation, rather than the widely held paradigm that the zona pellucida receptor is a single molecular entity. During these studies the use of blue native polyacrylamide gel electrophoresis, for the first time in mammalian sperm, has provided direct evidence that a number of multimeric zona receptor complexes indeed reside on the apical plasma membrane of capacitated sperm and that two of these complexes have the ability to interact with the zona pellucida. Proteomic analysis of these two complexes has indicated that molecular chaperones (CCT/TRiC complex and HSPD1) are responsible for the formation of each complex, and individually, these complexes contain a number of receptor proteins (ZPBP2, ZP3R and ADAMTS10) that potentially provide the zona pellucida affinity. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize

Characteristics of the Epididymal Luminal Environment Responsible for Sperm Maturation and Storage

The testicular spermatozoa of all mammalian species are considered functionally immature owing to their inability to swim in a progressive manner and engage in productive interactions with the cumulus–oocyte complex. The ability to express these key functional attributes develops progressively during the cells’ descent through the epididymis, a highly specialized ductal system that forms an integral part of the male reproductive tract. The functional maturation of the spermatozoon is achieved via continuous interactions with the epididymal luminal microenvironment and remarkably, occurs in the complete absence of de novo gene transcription or protein translation. Compositional analysis of the luminal fluids collected from the epididymis of a variety of species has revealed the complexity of this milieu, with a diversity of inorganic ions, proteins, and small non-coding RNA transcripts having been identified to date. Notably, both the quantitative and qualitative profile of each of these different luminal elements display substantial segment-to-segment variation, which in turn contribute to the regionalized functionality of this long tubule. Thus, spermatozoa acquire functional maturity in the proximal segments before being stored in a quiescent state in the distal segment in preparation for ejaculation. Such marked division of labor is achieved via the combined secretory and absorptive activity of the epithelial cells lining each segment. Here, we review our current understanding of the molecular mechanisms that exert influence over the unique intraluminal environment of the epididymis, with a particular focus on vesicle-dependent mechanisms that facilitate intercellular communication between the epididymal soma and maturing sperm cell population

The role of molecular chaperones in spermatogenesis and the post-testicular maturation of mammalian spermatozoa

Background: Spermatogenesis culminates in production of one of the most highly differentiated cells in biology, the spermatozoon. The gametes that emerge from the testes are, however, functionally immature and only acquire full functionality once they have completed a process of post-testicular maturation in the epididymis and female reproductive tract. Remarkably, this acquisition of sperm function occurs while these cells are transcriptionally and translationally silent and is therefore highly dependent on post-translational modifications to their existing protein complement. In this review, we consider the emerging roles of several prominent molecular chaperone families in orchestrating both the morphological differentiation of male germ cells during spermatogenesis and their functional transformation during sperm maturation. Methods: Journal databases were searched using key words, including chaperone, heat shock protein, testes, spermatogenesis, spermatozoa, epididymal maturation, capacitation and fertilization. Results: In the past two decades, molecular chaperones have been acknowledged to play key roles in controlling both the morphological transformation of germ cells during spermatogenesis and the post-testicular maturation of these cells as they transit the male and female reproductive tracts. Furthermore, there is mounting evidence that aberrant chaperone expression may be a major contributing factor to the defective sperm function seen in many cases of male infertility. Conclusions: Molecular chaperones are critically involved in all phases of sperm development. Targeted disruption of these proteins has the ability to arrest spermatogenesis, compromise sperm maturation and inhibit fertilization. These proteins therefore hold considerable promise as targets for novel contraceptive strategies and as diagnostic biomarkers for male infertility

The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition

One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI), recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2), as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF) success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function

Sperm-zona pellucida interaction: molecular mechanisms and the potential for contraceptive intervention

At the moment of insemination, millions of mammalian sperm cells are released into the female reproductive tract with the single goal of finding the oocyte. The spermatozoa subsequently ignore the thousands of cells they make contact with during their journey to the site of fertilization, until they reach the surface of the oocyte. At this point, they bind tenaciously to the acellular coat, known as the zona pellucida, which surrounds the oocyte and orchestrate a cascade of cellular interactions that culminate in fertilization. These exquisitely cell- and species- specific recognition events are among the most strategically important cellular interactions in biology. Understanding the cellular and molecular mechanisms that underpin them has implications for the etiology of human infertility and the development of novel targets for fertility regulation. Herein we describe our current understanding of the molecular basis of successful sperm–zona pellucida binding